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Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinson’s disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). In this advance, we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1. Description of files: Figure 1 Figure 1c OPA1 Coomassie: First gel: +Phos Tag Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Second gel: -Phos Tag Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Figure 1c blot: Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Figure 1d Coomassie: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1d autorad: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1f FLAG: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f HSP60: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f OPA1: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f pS228: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f PINK1 pS228 FLAG merge: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1g PINK1 pS228 PINK1 multiplex: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g pS228: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g PINK1: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g Tom20: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1h HSP60: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP), Rest of the lanes: Extra samples not included in the paper. Figure 1h OPA1: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1h pS228 PINK1: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1h pUb: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1- Figure Supplement 2b: Recombinant jason chin post-purification. 2 lanes loaded per construct, one for amylose purification and another for the subsequent 6His purification. Lane1 and Lane5 are included in the paper. Lane 1 - WT PINK1, amylose (included in paper), Lane 2 - WT PINK1, 6His, Lane 3 - KI PINK1, amylose, Lane 4 - KI PINK1, 6His, Lane 5 - pSer228 PINK1, amylose (included in paper), Lane 6 - pSer228 PINK1, 6His, Lane 7 - pSer284 PINK1, amylose, Lane 8 - pSer284 PINK1, 6His, Lane 9 - pSer402 PINK1, amylose, Lane 10 - pSer402 PINK1, 6His Figure 1- Figure Supplement 2c: Lane10 and Lane11 are included in the paper. Lane 1 - 2mg/ml BSA, Lane 2 - 1mg/ml BSA, Lane 3 - 0.5mg/ml BSA, Lane 4 - 0.25mg/ml BSA, Lane 5 - 0.125 mg/ml BSA, Lane 6 – ladder, Lane 7 - unrelated PINK1 construct (Ser228A), Lane 8 - unrelated PINK1 construct (Ser284A), Lane 9 - KI PINK1, Lane 10 - WT PINK1, Lane 11 - pSer228 PINK1, 6His, Lane 12 - pSer284 PINK1, 6His Figure 1- Figure Supplement 2d (+ phos tag): Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper) Figure 1- Figure Supplement 2d (no phos tag): Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper) Figure 1- Figure Supplement 2f Coomassie: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1 - Figure Supplement 2f autorad: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1 - Figure Supplement 3: Immunofluorescence images as mentioned in the paper Figure 2 Figure 2a FLAG: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2a HSP60: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP), rest of the lanes: Extra samples not included in the paper Figure 2a OPA1: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2a Phostag: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2- Figure Supplement 1a: Lane 1: Marker, Lane 3: emp 3-FLAG (-CCCP), Lane 4: emp 3-FLAG (+CCCP), Lane 5: KI PINK1 3-FLAG (-CCCP), Lane 6: KI PINK1 3-FLAG (+CCCP), Lane 7: WT PINK1 3-FLAG (-CCCP), Lane 8: WT PINK1 3-FLAG (+CCCP), Rest of the lanes: Not included in the paper Figure 2- Figure Supplement 1b: Lane 1: Marker, Lane3-5: 1% TX-100 (Total, soluble, insoluble), Lane 6-8: 1% SDS (Total, soluble, insoluble), Lane 10-12: 2% SDS (Total, soluble, insoluble) Figure 2- Figure Supplement 1c: Lane 1: Marker, Lane3-5: WT DMSO (Input, SN, IP), Lane 6-8: WT CCCP (Input, SN, IP), Lane 10-12: KI DMSO (Input, SN, IP), Lane 14-16: KI CCCP (Input, SN, IP) Figure 2- Figure Supplement 1d DCP: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ps228: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d mix: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d HSP60 KO: Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d HSP60 WT: Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ACSL1 HSP60 KO: HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ACSL1 HSP60 WT: HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1e: Lane 1: Marker, Lane 3: WT Trypsin (-CCCP), Lane 5: WT chymotrypsin (-CCCP), Lane 7: WT Trypsin (+CCCP), Lane 9: WT chymotrypsin (+CCCP), Lane 11: KI Trypsin (-CCCP), Lane 13: KI chymotrypsin (-CCCP), Lane 15: KI Trypsin (+CCCP), Lane 17: KI chymotrypsin (+CCCP) Figure 3 Figure 3c OPA1 PINK1: Upper blot: OPA1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Lower blot: PINK1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 3c ps228 PINK1: Upper blot: pS228 PINK1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Lower blot: HSP60 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 3c pUb: Blot: pS65 Ub Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 4 Figure 4e PINK1: Full blot (cut according to kDa before probing with antibody); Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+CCCP for 180 min), Lane 14: C412A PINK 3FLAG (-CCCP), Lane 15: C412A PINK 3FLAG (+CCCP for 15 min), Lane 16: C412A PINK 3FLAG (+CCCP for 30 min), Lane 17: C412A PINK 3FLAG (+CCCP for 45 min), Lane 18: C412A PINK 3FLAG (+CCCP for 60 min), Lane 19: C412A PINK 3FLAG (+CCCP for 120 min), Lane 20: C412A PINK 3FLAG (+CCCP for 180 min) Figure 4e GAPDH OPA: Upper bands: OPA1, Lower bands: GAPDH; Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+C

Leucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson’s disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα unexpectedly does not activate LRRK1 by phosphorylating the kinase domain, but instead phosphorylates a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain of the GTPase domain. These residues are positioned at the equivalent region of the LRRK2 DK helix reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 represents an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation of Ser1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ~3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 activates LRRK1 by promoting interaction and stabilization of the aC-helix on the kinase domain. This study provides new fundamental insights into the mechanism controlling LRRK1 activity and reveals a novel unexpected activation mechanism.

We reported and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in preclassified clinical specimens. The method uses a 15 min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration, and OpenSWATH software for data analysis. We applied the method to a cohort containing 204 FFPE tissue samples from 58 prostate cancer patients and 10 benign prostatic hyperplasia patients. Altogether we identified 27,975 proteotypic peptides and 4037 SwissProt proteins from these 204 samples. Compared to a reference SWATH method with a 2 h gradient, we found 3800 proteins were quantified by the two methods on two different instruments with relatively high consistency (r = 0.77). The accelerated method consumed only 17% instrument time, while quantifying 80% of proteins compared to the 2 h gradient SWATH. Although the missing value rate increased by 20%, batch effects reduced by 21%. 75 deregulated proteins measured by the accelerated method were selected for further validation. A shortlist of 134 selected peptide precursors from the 75 proteins were analyzed using MRM-HR, and the results exhibited high quantitative consistency with the 15 min SWATH method (r = 0.89) in the same sample set. We further verified the applicability of these 75 proteins in separating benign and malignant tissues (AUC = 0.99) in an independent prostate cancer cohort (n = 154). Altogether, the results showed that the 15 min gradient microflow SWATH accelerated large-scale data acquisition by 6 times, reduced batch effect by 21%, introduced 20% more missing values, and exhibited comparable ability to separate disease groups.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550. ispartof: JACS AU vol:1 issue:6 pages:750-765 ispartof: location:United States status: published

Ppp5 (protein phosphatase 5) is a serine/threonine protein phosphatase that has been conserved throughout eukaryotic evolution. In mammalian cells, FLAG-tagged Ppp5 and endogenous Ppp5 are found to interact with endogenous Hsp (heat-shock protein) 70, as well as Hsp90. Incubation of cells with arachidonic acid or the microtubule-depolymerizing agent, nocodazole, causes loss of interaction of Hsp70 and Hsp90 with FLAG-tagged Ppp5 and increase of Ppp5 activity. In response to the same treatments, endogenous Ppp5 undergoes proteolytic cleavage of the N- and C-termini, with the subsequent appearance of high-molecular-mass species. The results indicate that Ppp5 is activated by proteolysis on dissociation from Hsps, and is destroyed via the proteasome after ubiquitination. Cleavage at the C-terminus removes a nuclear localization sequence, allowing these active cleaved forms of Ppp5 to translocate to the cytoplasm. The response of Ppp5 to arachidonic acid and nocodazole suggests that Ppp5 may be required for stress-related processes that can sometimes cause cell-cycle arrest, and leads to the first description for in vivo regulation of Ppp5 activity.

We report and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in clinical specimens. The method uses 15-min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration and OpenSWATH software for data analysis.We applied the method to a cohort 204 of FFPE prostate tissue samples from 58 prostate cancer patients and 10 prostatic hyperplasia patients. Altogether we identified 27,976 proteotypic peptides and 4,043 SwissProt proteins from these 204 samples. Compared to a reference SWATH method with 2-hour gradient the accelerated method consumed only 27% instrument time, quantified 80% proteins and showed reduced batch effects. 3,800 proteins were quantified by both methods in two different instruments with relatively high consistency (r = 0.77). 75 proteins detected by the accelerated method with differential abundance between clinical groups were selected for further validation. A shortlist of 134 selected peptide precursors from the 75 proteins were analyzed using MRM-HR, exhibiting high quantitative consistency with the 15-min SWATH method (r = 0.89) in the same sample set. We further verified the capacity of these 75 proteins in separating benign and malignant tissues (AUC = 0.99) in an independent prostate cancer cohort (n=154).Overall our data show that the single-shot short gradient microflow-LC SWATH MS method achieved about 4-fold acceleration of data acquisition with reduced batch effect and a moderate level of protein attrition compared to a standard SWATH acquisition method. Finally, the results showed comparable ability to separate clinical groups.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a long-established EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 – but not of its homologues LPP1 or LPP2 – diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Melanoma cells can create and follow their own gradients of attractant, via a new mechanism by which tumour cells may undergo metastasis.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a long-established EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.

Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription.

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/β-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.

Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins-PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.

Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

Exo1 is a nuclease involved in mismatch repair, DSB repair, stalled replication fork processing and in the DNA damage response triggered by dysfunctional telomeres. In budding yeast and mice, Exo1 creates single-stranded DNA (ssDNA) at uncapped telomeres. This ssDNA accumulation activates the checkpoint response resulting in cell cycle arrest. Here, we demonstrate that Exo1 is phosphorylated when telomeres are uncapped in cdc13-1 and yku70Delta yeast cells, and in response to the induction of DNA damage. After telomere uncapping, Exo1 phosphorylation depends on components of the checkpoint machinery such as Rad24, Rad17, Rad9, Rad53 and Mec1, but is largely independent of Chk1, Tel1 and Dun1. Serines S372, S567, S587 and S692 of Exo1 were identified as targets for phosphorylation. Furthermore, mutation of these Exo1 residues altered the DNA damage response to uncapped telomeres and camptothecin treatment, in a manner that suggests Exo1 phosphorylation inhibits its activity. We propose that Rad53-dependent Exo1 phosphorylation is involved in a negative feedback loop to limit ssDNA accumulation and DNA damage checkpoint activation.

The protein kinases IRAK [IL-1 (interleukin 1) receptor-associated kinase] 1 and 4 play key roles in a signalling pathway by which bacterial infection or IL-1 trigger the production of inflammatory mediators. In the present study, we demonstrate that IRAK1 and IRAK4 phosphorylate Pellino isoforms in vitro and that phosphorylation greatly enhances Pellino's E3 ubiquitin ligase activity. We show that, in vitro, Pellino 1 can combine with the E2 conjugating complex Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) to catalyse the formation of K63-pUb (Lys63-linked polyubiquitin) chains, with UbcH3 to catalyse the formation of K48-pUb chains and with UbcH4, UbcH5a or UbcH5b to catalyse the formation of pUb-chains linked mainly via Lys11 and Lys48 of ubiquitin. In IRAK1-/- cells, the co-transfection of DNA encoding wild-type IRAK1 and Pellino 2, but not inactive mutants of these proteins, induces the formation of K63-pUb-IRAK1 and its interaction with the NEMO [NF-kappaB (nuclear factor kappaB) essential modifier] regulatory subunit of the IKK (inhibitor of NF-kappaB kinase) complex, a K63-pUb-binding protein. These studies suggest that Pellino isoforms may be the E3 ubiquitin ligases that mediate the IL-1-stimulated formation of K63-pUb-IRAK1 in cells, which may contribute to the activation of IKKbeta and the transcription factor NF-kappaB, as well as other signalling pathways dependent on IRAK1/4.

The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.

Recent studies have shown that PRC1-like Polycomb repressor complexes monoubiquity-late chromatin on histone H2A at lysine residue 119. Here we have analyzed the function of the polycomb protein Mel-18. Using affinity-tagged human MEL-18, we identify a polycomb-like complex, melPRC1, containing the core PRC1 proteins, RING1/2, HPH2, and CBX8. We show that, in ES cells, melPRC1 can functionally substitute for other PRC1-like complexes in Hox gene repression. A reconstituted subcomplex containing only Ring1B and Mel-18 functions as an efficient ubiquitin E3 ligase. This complex ubiquitylates free histone substrates nonspecifically but is highly specific for histone H2A lysine 119 in the context of nucleosomes. Mutational analysis demonstrates that while Ring1B is required for E3 function, Mel-18 directs this activity to H2A lysine 119 in chromatin. Moreover, this substrate-targeting function of Mel-18 is dependent on its prior phosphorylation at multiple residues, providing a direct link between chromatin modification and cell signaling pathways.

The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

Glycogen synthase kinase 3 (GSK3), a key component of the insulin and wnt signaling pathways, is unusual, as it is constitutively active and is inhibited in response to upstream signals. Kinase activity is thought to be increased by intramolecular phosphorylation of a tyrosine in the activation loop (Y216 in GSK3beta), whose timing and mechanism is undefined. We show that GSK3beta autophosphorylates Y216 as a chaperone-dependent transitional intermediate possessing intramolecular tyrosine kinase activity and displaying different sensitivity to small-molecule inhibitors compared to mature GSK3beta. After autophosphorylation, mature GSK3beta is then an intermolecular serine/threonine kinase no longer requiring a chaperone. This shows that autoactivating kinases have adopted different molecular mechanisms for autophosphorylation; and for kinases such as GSK3, inhibitors that affect only the transitional intermediate would be missed in conventional drug screens.

FRAT1, like its Xenopus homolog glycogen synthase kinase-3 (GSK-3)-binding protein, is known to inhibit GSK-3-mediated phosphorylation of beta-catenin. It is currently unknown how FRAT-GSK-3-binding protein activity toward GSK-3 is regulated. FRAT1 has recently been shown to be a phosphoprotein in vivo; however, the responsible kinase(s) have not been determined. In this study, we identified Ser188 as a phosphorylated residue in FRAT1. The identity of the kinase that catalyzes Ser188 phosphorylation and the significance of this phosphorylation to FRAT1 function were investigated. Protein kinase A (PKA) was found to phosphorylate Ser188 in vitro as well as in intact cells. Importantly, activation of endogenous cAMP-coupled beta-adrenergic receptors with norepinephrine stimulated the phosphorylation of FRAT1 at Ser188. GSK-3 was also able to phosphorylate FRAT1 at Ser188 and other residues in vitro or when overexpressed in intact cells. In contrast, endogenous GSK-3 did not lead to significant FRAT1 phosphorylation in cells, suggesting that GSK-3 is not a major FRAT1 kinase in vivo. Phosphorylation of Ser188 by PKA inhibited the ability of FRAT1 to activate beta-catenin-dependent transcription. In conclusion, PKA phosphorylates FRAT1 in vitro as well as in intact cells and may play a role in regulating the inhibitory activity of FRAT1 toward GSK-3.

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.

Udgivelsesdato: 2006-Jul Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through protein-protein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.

Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr167 and Ser171 is required for the activation of MELK. The expression of MELK activity also requires reducing agents such as dithiothreitol or reduced glutathione. Furthermore, we show that MELK is a Ca2+-binding protein and is inhibited by physiological Ca2+ concentrations. The smallest MELK fragment that was still catalytically active comprises the N-terminal catalytic domain and the flanking ubiquitin-associated domain. A C-terminal fragment of MELK functions as an autoinhibitory domain. Our data show that the activity of MELK is regulated in a complex manner and offer new perspectives for the further elucidation of its biological function.

Kaposi sarcoma-associated herpes virus (KSHV) encodes a D-like cyclin (K-cyclin) that is thought to contribute to the viral oncogenicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4 and 6, generating enzymes with a substrate selectivity deviant from CDK4 and CDK6 activated by D-type cyclins, suggesting different biochemical and biological functions. Here we report the identification of the actin- and calmodulin-binding protein caldesmon (CALD1) as a novel K-cyclin.CDK substrate, which is not phosphorylated by D.CDK. CALD1 plays a central role in the regulation of microfilament organization, consequently controlling cell shape, adhesion, cytokinesis and motility. K-cyclin.CDK6 specifically phosphorylates four Ser/Thr sites in the human CALD1 carboxyl terminus, abolishing CALD1 binding to its effector protein, actin, and its regulator protein, calmodulin. CALD1 is hyperphosphorylated in cells following K-cyclin expression and in KSHV-transformed lymphoma cells. Moreover, expression of exogenous K-cyclin results in microfilament loss and changes in cell morphology; both effects are reliant on CDK catalysis and can be reversed by the expression of a phosphorylation defective CALD1. Together, these data strongly suggest that K-cyclin expression modulates the activity of caldesmon and through this the microfilament functions in cells. These results establish a novel link between KSHV infection and the regulation of the actin cytoskeleton.

A substrate for PKBa (protein kinase Bα) was detected in liver extracts, and was purified and identified as CRHSP24 (calcium-regulated heat-stable protein of apparent molecular mass 24 kDa). PKBa, as well as SGK1 (serum- and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase), phosphorylated CRHSP24 stoichiometrically at Ser 5 2 in vitro and its brain-specific isoform PIPPin at the equivalent residue (Ser 5 8 ). CRHSP24 became phosphorylated at Ser 5 2 when HEK-293 (human embryonic kidney) cells were stimulated with IGF-1 (insulin-like growth factor-1) and this was prevented by inhibitors of PI3K (phosphoinositide 3-kinase), but not by rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] or PD 184352, an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade and hence the activation of RSK. IGF-1 induced a similarphosphorylation of CRHSP24 in ES (embryonic stem) cells from wild-type mice or mice that express the PDK I (3-phosphoinositide-dependent kinase I) mutant (PDK 1[L155E]) that activates PKBα normally, but cannot activate SGK. CRHSP24 also became phosphorylated at Ser 5 2 in response to EGF (epidermal growth factor) and this was prevented by blocking activation of both the classical MAPK cascade and the activation of PKBa, but not if just one of these pathways was inhibited. DYRK2 (dual-specificity tyrosine-phosphorylated and -regulated protein kinase 2) phosphorylated CRHSP24 at Ser 3 0 , Ser 3 2 and Ser 4 1 in vitro, and Ser 4 1 was identified as a site phosphorylated in cells. These and other results demonstrate that CRHSP24 is phosphorylated at Ser 5 2 by PKBa in response to IGF-1, at Ser 5 2 by PKBa and RSK in response to EGF, and at Ser 4 1 in the absence of IGF-1/EGF by a DYRK isofonn or another proline-directed protein kinase(s).

A protein expressed in immune cells and muscle was detected in muscle extracts as a substrate for several SAPKs (stress-activated protein kinases). It interacted specifically with the F-actin capping protein CapZ in splenocytes, and was therefore termed ‘CapZIP’ (CapZ-interacting protein). Human CapZIP was phosphorylated at Ser-179 and Ser-244 by MAPKAP-K2 (mitogen-activated protein kinase-activated protein kinase 2) or MAPKAP-K3 in vitro. Anisomycin induced the phosphorylation of CapZIP at Ser-179 in Jurkat cells, which was prevented by SB 203580, consistent with phosphorylation by MAPKAP-K2 and/or MAPKAP-K3. However, osmotic shock-induced phosphorylation of Ser-179 was unaffected by SB 203580. These and other results suggest that CapZIP is phosphorylated at Ser-179 in cells by MAPKAP-K2/MAPKAP-K3, and at least one other protein kinase. Stress-activated MAP kinase family members phosphorylated human CapZIP at many sites, including Ser-68, Ser-83, Ser-108 and Ser-216. Ser-108 became phosphorylated when Jurkat cells were exposed to osmotic shock, which was unaffected by SB 203580 and/or PD 184352, or in splenocytes from mice that do not express either SAPK3/p38γ or SAPK4/p38δ. Our results suggest that CapZIP may be phosphorylated by JNK (c-Jun N-terminal kinase), which phosphorylates CapZIP to >5 mol/mol within minutes in vitro. Osmotic shock or anisomycin triggered the dissociation of CapZIP from CapZ in Jurkat cells, suggesting that phosphorylation of CapZIP may regulate the ability of CapZ to remodel actin filament assembly in vivo.

The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. Here we identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. We show that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria.

Phosphatidylinositol transfer protein alpha (PITPalpha) participates in the supply of phosphatidylinositol ( PI) required for many cellular events including phospholipase C (PLC) beta and gamma signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPalpha at Ser(166) resulted in inhibition of receptor-stimulated PLC activity. Ser(166) is localized in a small pocket between the 165 - 172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPalpha. To allow phosphorylation at Ser(166), a distinct structural form is postulated, and mutation of Thr(59) to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPalpha from rat brain or in vitro phosphorylated PITPalpha demonstrated that phosphorylated PITPalpha is structurally distinct from the non-phosphorylated form. Phosphorylated PITPalpha was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPalpha structure has to relax to allow access to the Ser(166) site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling.

Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts. Microcystin-Sepharose chromatography and mass spectrometry were utilized to affinity purify and identify protein phosphatase-associated proteins from isolated rat liver nuclei. Far Western analysis with labeled protein phosphatase 1 (PP1) showed that many more PP1 binding proteins exist in the nucleus than were previously demonstrated. Mass spectrometry confirmed the presence in the nucleus of the mammalian PP1 isoforms alpha1, alpha2, beta, and gamma1, plus the Aalpha and several of the B and B' subunits that are complexed to PP2A. Other proteins enriched on the microcystin matrix include the spliceosomal proteins known as the U2 snRNPs SAP145 and SAP155 and the U5 snRNPs p116 and p200, myosin heavy chain, and a nuclear PP1 myosin-targeting subunit related to M110. The putative RNA binding protein ZAP was also established as a nuclear PP1 binding protein using the criteria of co-purification with PP1 on microcystin-Sepharose, co-immunoprecipation, binding PP1 in an overlay assay, and presence of a putative PP1 binding site (KKRVRWAD). These results further support a key role for protein phosphatases in several nuclear functions, including the regulation of pre-mRNA splicing.

Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCdelta and PKCtheta correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCdelta, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCdelta or wild-type PKCdelta followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCdelta was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1-glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCdelta acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCdelta on at least 18 sites. The importance of three of the PKCdelta phosphorylation sites in IRS-1 was shown in vitro by a 75-80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCdelta on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCdelta modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1−/− ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1L155E/L155E knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction1. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras–Raf–MEK–ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation2,3,4,5, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.

Protein phosphatase 4 (PPP4) is a ubiquitous essential protein serine/threonine phosphatase found in higher eukaryotes. Coordinate variation of the levels of the catalytic subunit (PPP4c) and the regulatory subunit (R2)suggests that PPP4c and R2 form a heterodimeric core to which other regulatory subunits bind. Two proteins that specifically co-purify with Flag-epitope-tagged R2 expressed in HEK-293 cells were identified as Gemin3 and Gemin4. These two proteins have been identified previously as components of the Survival of Motor Neurons (SMN) protein complex, which is functionally defective in the hereditary disorder spinal muscular atrophy. Immuno-sedimentation of the epitope-tagged SMN protein complex from HeLa cells expressing CFP-SMN showed that the SMN protein interacts, as previously reported, with Gemin2 (SIP1), Gemin3 and Gemin4 and in addition associates with PPP4c. The SMN complex has been implicated in the assembly and maturation of small nuclear ribonucleoproteins (snRNPs). Expression of GFP-R2–PPP4c in HeLa cells enhances the temporal localisation of newly formed snRNPs, which is consistent with an association of R2-PPP4c with the SMN protein complex.

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.

Lipopolysaccharide (LPS) stimulates production of inflammatory mediators, partly by stabilizing [interleukin-6 (IL-6), cyclooxygenase 2 (COX-2)] and/or stimulating translation [tumour necrosis factor-alpha (TNF-alpha)] of their mRNAs. Such regulation depends on AU-rich elements (AREs) within the 3'-untranslated regions and is partially suppressed by SB 203580 (which inhibits SAPK2a/p38). The LPS-induced production of TNF-alpha and IL-6 is suppressed in MAPKAP-K2-deficient mice (a kinase activated by SAPK2a/p38). Here, we identify 18 macrophage proteins that bind to AREs and show that hnRNP A0 is a major substrate for MAPKAP-K2 in this fraction. MAPKAP-K2 phosphorylated hnRNP A0 at Ser84 in vitro and this residue became phosphorylated in LPS-stimulated cells. Phosphorylation was prevented by SB 203580 and suppressed in macrophages derived from MAPKAP-K2-deficient mice. The mRNAs encoding TNF-alpha, COX-2 and macrophage inflammatory protein-2 (MIP-2) bound to hnRNP A0 in LPS-stimulated macrophages, an interaction prevented by SB 203580. The LPS-induced stabilization of MIP-2 mRNA and production of MIP-2 protein were abolished when macrophages were incubated with SB 203580 plus PD 184352 (which inhibits the classical MAP kinase cascade). Our data suggest that LPS-induced binding of hnRNP A0 to AREs may contribute to the post-transcriptional regulation of specific mRNAs.

Eukaryotic elongation factor 2 (eEF2) kinase, the enzyme that inactivates eEF2, is controlled by phosphorylation. Previous work showed that stress-activated protein kinase 4 (SAPK4, also called p38delta) inhibits eEF2 kinase in vitro by phosphorylating Ser-359, while ribosomal protein S6 kinases inhibit eEF2 kinase by phosphorylating Ser-366 [Knebel, Morrice and Cohen (2001) EMBO J. 20, 4360-4369; Wang, Li, Williams, Terada, Alessi and Proud (2001) EMBO J. 20, 4370-4379]. In the present study we have examined the effects of the protein synthesis inhibitor anisomycin and tumour necrosis factor-alpha (TNF-alpha) on the phosphorylation of eEF2 kinase. We demonstrate that Ser-359, Ser-366 and two novel sites (Ser-377 and Ser-396) are all phosphorylated in human epithelial KB cells, but only the phosphorylation of Ser-359 and Ser-377 increases in response to these agonists and correlates with the dephosphorylation (activation) of eEF2. Ser-377 is probably a substrate of MAPKAP-K2/K3 (mitogen-activated protein kinase-activated protein kinase 2/kinase 3) in cells, because eEF2 kinase is phosphorylated efficiently by these protein kinases in vitro and phosphorylation of this site, induced by TNF-alpha and low (but not high) concentrations of anisomycin, is prevented by SB 203580, which inhibits SAPK2a/p38, their "upstream" activator. The phosphorylation of Ser-359 induced by high concentrations of anisomycin is probably catalysed by SAPK4/p38delta in cells, because no other stress-activated, proline-directed protein kinase tested phosphorylates this site in vitro and phosphorylation is insensitive to SB 203580. Interestingly, the phosphorylation of Ser-359 induced by TNF-alpha or low concentrations of anisomycin is suppressed by SB 203580, indicating that phosphorylation is also mediated by a novel pathway. Since the phosphorylation of Ser-377 does not inhibit eEF2 kinase in vitro, our results suggest that anisomycin or TNF-alpha inhibit eEF2 kinase via the phosphorylation of Ser-359.

The AGC family of protein kinases, which includes isoforms of protein kinase B (also known as Akt), ribosomal S6 protein kinase (S6K), and serum- and glucocorticoid-induced protein kinase (SGK) are activated in response to many extracellular signals and play key roles in regulating diverse cellular processes. They are activated by the phosphorylation of the T loop of their kinase domain by the 3-phosphoinositide-dependent protein kinase-1 and by phosphorylation of a residue located C-terminal to the kinase domain in a region termed the hydrophobic motif. Recent work has implicated the NIMA (never in mitosis, gene A)-related kinase-6 (NEK6) as the enzyme that phosphorylates the hydrophobic motif of S6K1 in vivo. Here we demonstrate that in addition to phosphorylating S6K1 and SGK1 at their hydrophobic motif, NEK6 also phosphorylates S6K1 at two other sites and phosphorylates SGK1 at one other site in vitro. Employing the Jerini pepSTAR method in combination with kinetic analysis of phosphorylation of variant peptides, we establish the key substrate specificity determinants for NEK6. Our analysis indicates that NEK6 has a strong preference for Leu 3 residues N-terminal to the site of phosphorylation. Its mutation to either Ile or Val severely reduced the efficacy with which NEK6-phosphorylated peptide substrates, and moreover, mutation of the equivalent Leu residue in S6K1 or SGK1 prevented phosphorylation of their hydrophobic motifs by NEK6 in vitro. However, these mutants of S6K1 or SGK1 still became phosphorylated at their hydrophobic motif following insulin-like growth factor-1 stimulation of transfected 293 cells. This study provides the first description of the basis for the substrate specificity of NEK6 and indicates that NEK6 is unlikely to be responsible for the IGF1-induced phosphorylation of the hydrophobic motif of S6K, SGK, and protein kinase B isoforms in vivo.

A protein phosphorylated efficiently in vitro by MAP kinase-activated protein kinase-2 (MAPKAP-K2) was purified from skeletal muscle extracts and identified as the calcium/calmodulin-dependent myosin light chain kinase (MLCK). The phosphorylation site was mapped to Ser 161 , a residue shown previously to be autophosphorylated by MLCK. The residue equivalent to Ser 161 became phosphorylated in vivo when rat hindlimbs were stimulated electrically. However, phosphorylation was triggered within seconds, whereas activation of MAPKAP-K2 required several minutes. Moreover, contraction-induced Ser 161 phosphorylation was similar in wild-type or MAPKAP-K2−/− mice. These results indicate that contraction-induced phosphorylation is probably catalyzed by MLCK and not MAPKAP-K2. Ser 161 phosphorylation induced the binding of MLCK to 14-3-3 proteins, but did not detectably affect the kinetic properties of MLCK. The sequence surrounding Ser 161 is unusual in that residue 158 is histidine. Previously, an arginine located three residues N-terminal to the site of phosphorylation was thought to be critical for the specificity of MAPKAP-K2.

Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM.

Exercise is known to increase insulin sensitivity and is an effective form of treatment for the hyperglycemia observed in type 2 diabetes. Activation of 5'-AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide riboside (AICAR), exercise, or electrically stimulated contraction leads to increased glucose transport in skeletal muscle. Here we report the first evidence of a direct interaction between AMPK and the most upstream component of the insulin-signaling cascade, insulin receptor substrate-1 (IRS-1). We find that AMPK rapidly phosphorylates IRS-1 on Ser-789 in cell-free assays as well as in mouse C2C12 myotubes incubated with AICAR. In the C2C12 myotubes activation of AMPK by AICAR matched the phosphorylation of IRS-1 on Ser-789. This phosphorylation correlates with a 65% increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity in C2C12 myotubes preincubated with AICAR. The binding of phosphatidylinositol 3-kinase to IRS-1 was not affected by AICAR. These results demonstrate the existence of an interaction between AMPK and early insulin signaling that could be of importance to our understanding of the potentiating effects of exercise on insulin signaling.

We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we identified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinase (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p38delta, but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta phosphorylated eEF2K at Ser359 in vitro, causing its inactivation. eEF2K became phosphorylated at Ser359 and its substrate eEF2 became dephosphorylated (activated) when KB cells were exposed to anisomycin, an agonist that activates all SAPKs, including SAPK4/p38delta. The anisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Ser359 was also induced by insulin-like growth factor-1. However, this was blocked by rapamycin, indicating that Ser359 is targeted by at least two signalling pathways.

Heat shock factor 1 (HSF1) is a serine-rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA-binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild-type HSF1 when expressed in hsf1(-/-) cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.