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Peutz-Jeghers syndrome is an inherited cancer syndrome that results in a greatly increased risk of developing tumors in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which LKB1 is regulated in cells is not known. Here, we demonstrate that stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser(431). We present pharmacological and genetic evidence that p90(RSK) mediated this phosphorylation in response to agonists that activate ERK1/2 and that cAMP-dependent protein kinase mediated this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and we demonstrate that full-length LKB1 expressed in 293 cells was prenylated by addition of a farnesyl group to Cys(433). Our data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.

Stress-activated protein kinase 1 (SAPK1), also called c-Jun N-terminal kinase (JNK), becomes activated in vivo in response to pro-inflammatory cytokines or cellular stresses. Its full activation requires the phosphorylation of a threonine and a tyrosine residue in a Thr-Pro-Tyr motif, which can be catalysed by the protein kinases mitogen-activated protein kinase kinase (MKK)4 and MKK7. Here we report that MKK4 shows a striking preference for the tyrosine residue (Tyr-185), and MKK7 a striking preference for the threonine residue (Thr-183) in three SAPK1/JNK1 isoforms tested (JNK1 alpha 1, JNK2 alpha 2 and JNK3 alpha 1). For this reason, MKK4 and MKK7 together produce a synergistic increase in the activity of each SAPK1/JNK isoform in vitro. The MKK7 beta variant, which is several hundred-fold more efficient in activating all three SAPK1/JNK isoforms than is MKK7 alpha', is equally specific for Thr-183. MKK7 also phosphorylates JNK2 alpha 2 at Thr-404 and Ser-407 in vitro, Ser-407 being phosphorylated much more rapidly than Thr-183 in vitro. Thr-404/Ser-407 are phosphorylated in unstimulated human KB cells and HEK-293 cells, and phosphorylation is increased in response to an osmotic stress (0.5 M sorbitol). However, in contrast with Thr-183 and Tyr-185, the phosphorylation of Thr-404 and Ser-407 is not increased in response to other agonists that activate MKK7 and SAPK1/JNK, suggesting that phosphorylation of these residues is catalysed by another protein kinase, such as CK2, which also phosphorylates Thr-404 and Ser-407 in vitro. MKK3, MKK4 and MKK6 all show a strong preference for phosphorylation of the tyrosine residue of the Thr-Gly-Tyr motifs in their known substrates SAPK2a/p38, SAPK3/p38 gamma and SAPK4/p38 delta. MKK7 also phosphorylates SAPK2a/p38 at a low rate (but not SAPK3/p38 gamma or SAPK4/p38 delta), and phosphorylation occurs exclusively at the tyrosine residue, demonstrating that MKK7 is intrinsically a 'dual-specific' protein kinase.

We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr139. Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr139, and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P3 or PtdIns(3,4)P2, is not phosphorylated at Tyr139 following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr139 of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr139in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P3, and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr139. These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr139 by a Src-family tyrosine kinase.

The interaction of BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) with Bcl-2/Bcl-XL is thought to neutralize the anti-apoptotic effects of the latter proteins, and may represent one of the mechanisms by which BAD promotes apoptosis. A variety of survival signals are reported to induce the phosphorylation of BAD at Ser112 or Ser136, triggering its dissociation from Bcl-2/Bcl-XL. Ser136 is thought to be phosphorylated by protein kinase B (PKB, also called Akt), which is activated when cells are exposed to agonists that stimulate phosphatidylinositol 3-kinase (PI3K). In contrast, Ser112 is reported to be phosphorylated by mitogen-activated protein (MAP) kinase-activated protein kinase-1 (MAPKAP-K1, also called RSK) and by cAMP-dependent protein kinase (PKA). Here we identify Ser155 as a third phosphorylation site on BAD. We find that Ser155 is phosphorylated preferentially by PKA in vitro and is the only residue in BAD that becomes phosphorylated when cells are exposed to cAMP-elevating agents. The phosphorylation of BAD at Ser155 prevents it from binding to Bcl-XL and promotes its interaction with 14-3-3 proteins. We also provide further evidence that MAPKAP-K1 mediates the phosphorylation of Ser112 in response to agonists that activate the classical MAP kinase pathway. However insulin-like growth factor 1, a potent activator of PI3K and PKB does not increase the phosphorylation of Ser136 in BAD-transfected HEK-293 cells, and nor is the basal level of Ser136 phosphorylation suppressed by inhibitors of PI3K.

Protein phosphatase 4 (PPP4) is a protein serine/threonine phosphatase that has been implicated in microtubule organization at centrosomes. Complexes of PPP4 with high apparent molecular masses (450 and 600 kDa) were purified from mammalian skeletal muscle and testis to near homogeneity. Amino acid sequences derived from a protein component present in both complexes were utilized to identify a human cDNA. The encoded putative PPP4 regulatory subunit (termed PPP4R2), comprising 453 amino acids, had a molecular mass of 50.4 kDa. The interaction of PPP4R2 with PPP4 catalytic subunit (PPP4c) was confirmed by co-sedimentation of PPP4c with PPP4R2 expressed in bacteria and human cells. PPP4c formed a complex of 450 kDa with baculovirus expressed His6-tagged PPP4R2. Immunocytological detection of PPP4R2 at centrosomes suggests that it may target PPP4c to this location. Native 450 kDa and 600 kDa PPP4 complexes are inactive, but can be activated by basic proteins, suggesting that PPP4R2 may also regulate the activity of PPP4c at centrosomal microtubule organising centres.

The p53-inducible gene product p21(WAF1/CIP1) plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed in Sf9 cells is subject to reversible phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr(145) or Ser(146). A phospho-specific antibody was developed that only bound to full-length p21 protein after phosphorylation in vitro at Ser(146), and this reagent was further used to demonstrate that the inactive isoform of p21 recovered from Sf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser(146). These data identify the first phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivo modulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.

Background: Clathrin-coated pits are formed at the plasma membrane by the assembly of the coat components, namely clathrin and adaptors from the cytosol. Little is known about the regulation and mechanism of this assembly process. Results: We have used an in vitro assay for clathrin-coated pit assembly to identify a novel component required for the invagination of newly formed coated pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new coated pits at the cell surface in permeabilised cells, the Rab5–GDI complex is required for ligand sequestration into clathrin-coated pits. Conclusions: We have identified Rab5 as a critical cytosolic component required for clathrin-coated pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.

Mitogen-activated protein kinase-activated protein kinase-1 (MAPKAP-K1; also known as p90 rsk ) contains two protein kinase domains in a single polypeptide. The N-terminal kinase domain is necessary for the phosphorylation of peptide substrates, whereas the C-terminal kinase domain is required for full activation of the N-terminal domain. Here we identify six sites in MAPKAP-K1a that become phosphorylated in transfected COS-1 cells. The inactive form of MAPKAP-K1a in unstimulated cells is partially phosphorylated at Ser222 and Ser733. Stimulation with phorbol 12-myristate 13-acetate induces the phosphorylation of Thr360, Ser364, Thr574, and Ser381 and increases the phosphorylation of Ser222 and Ser733. Our data indicate that mitogen-activated protein kinase activates the C-terminal kinase domain by phosphorylating Thr574 and contributes to the activation of the N-terminal kinase domain by phosphorylating Ser364. The activated C-terminal domain phosphorylates Ser381, which, together with phosphorylation of Ser364, activates the N-terminal kinase domain. The phosphorylation of Ser222and Ser733, which can be catalyzed by the N-terminal domain, does not activate MAPKAP-K1a per se, but Ser222 phosphorylation appears to be required for activation. Ser222, Ser364, and Ser381 are situated in analogous positions to phosphorylation sites in protein kinase B, protein kinase C, and p70S6K, suggesting a common mechanism of activation for these growth factor-stimulated protein kinases.

14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 zeta on Thr-233. This protein kinase has been identified as casein kinase Ialpha (CKIalpha) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 tau possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 zeta is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 zeta associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 zeta at the CKIalpha site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.

Background: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ). Results: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p 13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB α only in the presence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . Overexpression of PDK1 in 293 cells activated PKB α and potentiated the IGF1-induced phosphorylation of PKB α at Thr308. Experiments in which the PH domains of either PDK1 or PKB α were deleted indicated that the binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 to PKB α is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. Conclusions: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 in the activation of PKB α is at least partly substrate directed.

RepoMan is a protein phosphatase 1 (PP1) regulatory subunit that targets the phosphatase to key substrates throughout the cell cycle. Most work to date has focused on the mitotic roles of RepoMan/PP1, although equally important interphase role(s) have been demonstrated. Initial mapping of the interactome of nuclear RepoMan, both endogenous and tagged, was complicated by various factors, including antibody cross-reactivity and low sensitivity of the detection of chromatin-associated partners above the high background of proteins that bind nonspecifically to affinity matrices. We therefore adapted the powerful combination of fluorescence imaging with labeling-based quantitative proteomics to map the "fragmentomes" of specific regions of RepoMan. These regions demonstrate distinct localization patterns and turnover dynamics that reflect underlying binding events. The increased sensitivity and signal-to-noise ratio provided by this unique approach facilitated identification of a large number of novel RepoMan interactors, several of which were rigorously validated in follow-up experiments, including the association of RepoMan/PP1 with a specific PP2A-B56γ complex, interaction with ribosomal proteins and import factors involved in their nucleocytoplasmic transport and interaction with proteins involved in the response to DNA damage. This same strategy can be used to investigate the cellular roles of other modular proteins.

Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.

The form of protein phosphatase-1 associated with hepatic glycogen (PP1G) was purified to near homogeneity from rat liver by affinity chromatography on microcystin-Sepharose and gel-filtration. The enzyme is a heterodimer consisting of the catalytic subunit of PP1 (the α and β isoforms) complexed to a 33 kDa glycogen-binding (GL) subunit. The GL subunit binds phosphorylase a with high affinity, and is responsible for the enhanced dephosphorylation of glycogen synthase by PP1G and its allosteric inhibition by phosphorylase a.

Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.

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.

Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5′-DNA kinase/3′-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.

To identify new potential substrates for the MAP kinase signal-integrating kinases (Mnks), we employed a proteomic approach. The Mnks are targeted to the translational machinery through their interaction with the cap-binding initiation factor complex. We tested whether proteins retained on cap resin were substrates for the Mnks in vitro, and identified one such protein as PSF (the PTB (polypyrimidine tract-binding protein)-associated splicing factor). Mnks phosphorylate PSF at two sites in vitro, and our data show that PSF is an Mnk substrate in vivo. We also demonstrate that PSF, together with its partner, p54(nrb), binds RNAs that contain AU-rich elements (AREs), such as those for proinflammatory cytokines (e.g. tumor necrosis factor alpha (TNFalpha)). Indeed, PSF associates specifically with the TNFalpha mRNA in living cells. PSF is phosphorylated at two sites by the Mnks. Our data show that Mnk-mediated phosphorylation increases the binding of PSF to the TNFalpha mRNA in living cells. These findings identify a novel Mnk substrate. They also suggest that the Mnk-catalyzed phosphorylation of PSF may regulate the fate of specific mRNAs by modulating their binding to PSF.p54(nrb).

The spindle checkpoint delays anaphase onset until all chromosomes are correctly attached to microtubules. Ipl1 protein kinase (Aurora B) is required to correct inappropriate kinetochore–microtubule attachments and for the response to lack of tension between sister kinetochores. Here we identify residues in the checkpoint protein Mad3p that are phosphorylated by Ipl1p. When phosphorylation of Mad3p at two sites is prevented, the cell’s response to reduced kinetochore tension is dramatically curtailed. Our data provide strong evidence for a distinct checkpoint pathway responding to lack of sister kinetochore tension, in which Ipl1p-dependent phosphorylation of Mad3p is a key step.

The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, we used full-length dDYRK2 (Drosophila DYRK2) as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library. Of 14 independent dDYRK2 interacting clones identified, three were derived from the chromatin remodelling factor, SNR1 (Snf5-related 1), and three from the essential chromatin component, TRX (trithorax). The association of dDYRK2 with SNR1 and TRX was confirmed by co-immunoprecipitation studies. Deletion analysis showed that the C-terminus of dDYRK2 modulated the interaction with SNR1 and TRX. DYRK family member MNB (Minibrain) was also found to co-precipitate with SNR1 and TRX, associations that did not require the C-terminus of the molecule. dDYRK2 and MNB were also found to phosphorylate SNR1 at Thr102in vitro and in vivo. This phosphorylation required the highly conserved DH-box (DYRK homology box) of dDYRK2, whereas the DH-box was not essential for phosphorylation by MNB. This is the first instance of phosphorylation of SNR1 or any of its homologues and implicates the DYRK family of kinases with a role in chromatin remodelling.

The Ku70/80 heterodimer is a major player in non-homologous end joining and the repair of DNA double-strand breaks. Studies suggest that once bound to a DNA double-strand break, Ku recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form the DNA-dependent protein kinase holoenzyme complex (DNA-PK). We previously identified four DNA-PK phosphorylation sites on the Ku70/80 heterodimer: serine 6 of Ku70, serine 577 and 580 and threonine 715 of Ku80. This raised the interesting possibility that DNA-PK-dependent phosphorylation of Ku could provide a mechanism for the regulation of non-homologous end joining. Here, using mass spectrometry and phosphospecific antibodies we confirm that these sites are phosphorylated in vitro by purified DNA-PK. However, we show that neither DNA-PK nor the related protein kinase ataxia-telangiectasia mutated (ATM) is required for phosphorylation of Ku at these sites in vivo. Furthermore, Ku containing serine/threonine to alanine mutations at these sites was fully able to complement the radiation sensitivity of Ku negative mammalian cells indicating that phosphorylation at these sites is not required for non-homologous end joining. Interestingly, both Ku70 and Ku80 were phosphorylated in cells treated with the protein phosphatase inhibitor okadaic acid under conditions known to inactivate protein phosphatase 2A-like protein phosphatases. Moreover, okadaic acid-induced phosphorylation of Ku80 was inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. These results suggest that the phosphorylation of Ku70 and Ku80 is regulated by a protein phosphatase 2A-like protein phosphatase and a staurosporine sensitive protein kinase in vivo, but that DNA-PK-mediated phosphorylation of Ku is not required for DNA double-strand break repair.

Posttranscriptional regulatory mechanisms control TNFalpha expression through AU-rich elements in the 3'UTR of its mRNA. This is mediated through Erk and p38 MAP kinase signaling, although the mechanisms involved remain poorly understood. Here, we show that the MAP kinase signal-integrating kinases (Mnks), which are activated by both these pathways, regulate TNFalpha expression in T cells via the 3'UTR. A selective Mnk inhibitor or siRNA-mediated knockdown of Mnk1 inhibits TNFalpha production in T cells, whereas Mnk1 overexpression enhances expression of a reporter construct containing the TNFalpha 3'UTR. We identify ARE binding proteins that are Mnk substrates, such as hnRNP A1, which they phosphorylate at two sites in vitro. hnRNP A1 is phosphorylated in response to T cell activation, and this is blocked by Mnk inhibition. Moreover, Mnk-mediated phosphorylation decreases binding of hnRNP A1 to TNFalpha-ARE in vitro or TNFalpha-mRNA in vivo. Therefore, Mnks are novel players in cytokine regulation and potential new targets for anti-inflammatory therapy.

14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3–Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-d-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a Ser/Thr kinase with an essential role in insulin and growth-factor signalling. PDK1 activity towards protein kinase B (PKB) is partially regulated by its pleckstrin homology (PH) domain, which preferentially binds to 3-phosphoinositides. However, the precise molecular mechanism of this regulation is not well understood. Here, the cloning, purification and crystallization of a 150-amino-acid C-terminal region of PDK1 containing the PH domain is reported. A crystal of the PDK1 PH domain grown in the presence of inositol 1,3,4,5-tetrakisphosphate and derivatized with AuCN diffracted to 1.5 A at a synchrotron source. Diffraction data collected near the Au edge resulted in an anomalous Patterson map with a 30sigma peak.

Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 min of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the mitogen-activated protein kinase kinase (MEK) inhibitor PD098059 (2′-amino-3′-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H2O2resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H2O2induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PD098059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEKCA), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated cells, tropomyosin-1 was found diffuse in the cells, whereas it quickly colocalized with actin and stress fibers upon stimulation of ERK by H2O2or by expression of MEKCA. We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7 (1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated downstream of ERK, an event that modulates its interaction with actin, may lead to further understanding of the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.

MKK5 expressed as a glutathione S-transferase fusion protein in human embryonic kidney 293 cells activated full-length extracellular-signal-regulated protein kinase (ERK)5 (ERK5wt) as well as the isolated catalytic domain (ERK5cat) in vitro. Activation was accompanied by the phosphorylation of Thr(219) and Tyr(221), the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thr(219), but not Tyr(221), possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyr(221) alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thr(28), Ser(421), Ser(433), Ser(496), Ser(731) and Thr(733). ERK5 phosphorylated at Thr(219), but not Tyr(221), phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thr(219) and Tyr(221). Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Ser(129), Ser(137), Ser(142) and Ser(149), which are located within the region in MKK5 that is thought to interact with ERK5.

The serine/threonine protein kinase LKB1 functions as a tumour suppressor, and mutations in this enzyme lead to the inherited Peutz—Jeghers cancer syndrome. We previously found that LKB1 was phosphorylated at Thr-366 in vivo, a residue conserved in mammalian, Xenopus and Drosophila LKB1, located on a C-terminal non-catalytic moiety of the enzyme. Mutation of Thr-366 to Ala or Asp partially inhibited the ability of LKB1 to suppress growth of G361 melanoma cells, but did not affect LKB1 activity in vitro or LKB1 localization in vivo. As a first step in exploring the role of this phosphorylation further, we have generated a phosphospecific antibody specifically recognizing LKB1 phosphorylated at Thr-366 and demonstrate that exposure of cells to ionizing radiation (IR) induced a marked phosphorylation of LKB1 at Thr-366 in the nucleus. Thr-366 lies in an optimal phosphorylation motif for the phosphoinositide 3-kinase-like kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia-related kinase (ATR), which function as sensors for DNA damage in cells and mediate cellular responses to DNA damage. We demonstrate that both DNA-PK and ATM efficiently phosphorylate LKB1 at Thr-366 in vitro and provide evidence that ATM mediates this phosphorylation in vivo. This is based on the finding that LKB1 is not phosphorylated in a cell line lacking ATM in response to IR, and that agents which induce cellular responses via ATR in preference to ATM poorly induce phosphorylation of LKB1 at Thr-366. These observations provide the first link between ATM and LKB1 and suggest that ATM could regulate LKB1.

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.

Peutz-Jeghers syndrome is an inherited cancer syndrome, which results in a greatly increased risk of developing tumours in those affected. The causative gene encodes a nuclear-localized protein kinase, termed LKB1, which is predicted to function as a tumour suppressor. The mechanism by which LKB1 is regulated in cells is not known, and nor have any of its physiological substrates been identified. Recent studies have demonstrated that LKB1 is phosphorylated in cells. As a first step towards identifying the roles that phosphorylation of LKB1 play, we have mapped the residues that are phosphorylated in human embryonic kidney (HEK)-293 cells, as well as the major in vitro autophosphorylation sites. We demonstrate that LKB1 expressed in HEK-293 cells, in addition to being phosphorylated at Ser(431), a previously characterized phosphorylation site, is also phosphorylated at Ser(31), Ser(325) and Thr(366). Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr(336) as well as at Thr(366). We were unable to detect autophosphorylation at Thr(189), a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser(31) and Ser(325) in HEK-293 cells to the same extent as the wild-type enzyme, indicating that LKB1 does not phosphorylate itself at these residues. We show that phosphorylation of LKB1 does not directly affect its nuclear localization or its catalytic activity in vitro, but that its phosphorylation at Thr(336), and perhaps to a lesser extent at Thr(366), inhibits LKB1 from suppressing cell growth.

The CD11/CD18 (beta(2)) integrins are leukocyte-specific adhesion receptors, and their ability to bind ligands on other cells can be activated by extracellular stimuli. During cell activation, the CD18 chain is known to become phosphorylated on serine and functionally important threonine residues located in the intracellular C-terminal tail. Here, we identify catalytic domain fragments of protein kinase C (PKC) delta and PKCbetaI/II as the major protein kinases in leukocyte extracts that phosphorylate a peptide corresponding to the cytoplasmic tail of the integrin CD18 chain. The sites phosphorylated in vitro were identified as Ser-745 and Thr-758. PKCalpha and PKCeta also phosphorylated these residues, and PKCalpha additionally phosphorylated Thr-760. Ser-745, a novel site, was shown to become phosphorylated in T cells in response to phorbol ester stimulation. Ser-756, a residue not phosphorylated by PKC isoforms, also became phosphorylated in T cells after phorbol ester stimulation. When leukocyte extracts were subjected to affinity chromatography on agarose to which residues 751-761 of the CD18 chain phosphorylated at Thr-758 were bound covalently, the only proteins that bound specifically were identified as isoforms of 14-3-3 proteins. Thus, PKC-mediated phosphorylation of CD18 after cell stimulation could lead to the recruitment of 14-3-3 proteins to the activated integrin, which may play a role in regulating its adhesive state or ability to signal.

Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr(24), Ser(256) and Ser(319)) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser(329)) as a novel in vivo phosphorylation site on FKHR. Ser(329) phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser(329) is phosphorylated in unstimulated HEK-293 cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser(329) specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser(329) in vivo.

ATR is a large, >300 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with the DNA-PK, are part of a new family of PI-3 kinase related proteins. All members of this family play important roles in checkpoints which operate to permit cell survival following many forms of DNA damage. We have expressed ATR protein in HEK293 cells and purified the protein to near-homogeneity. We show that pure ATR is a protein kinase which is activated by circular single-stranded, double-stranded or linear DNA. Thus ATR is a new member of a sub-family of PIK related kinases, founded by the DNA-PK, which are activated in the presence of DNA. Unlike DNA-PK, ATR does not appear to require Ku proteins for its activation by DNA. We show directly that, like ATM and DNA-PK, ATR phosphorylates the genome surveillance protein p53 on serine 15, a site which is up-regulated in response to DNA damage. In addition, we find that ATR has a substrate specificity similar to, but unique from, the DNA-PK in vitro, suggesting that these proteins have overlapping but distinct functions in vivo. Finally, we find that the kinase activity of ATR in the presence and absence of DNA is suppressed by caffeine, a compound which is known to induce loss of checkpoint control. Our results are consistent with the notion that ATR plays a role in monitoring DNA structure and phosphorylation of proteins involved in the DNA damage response pathways.

The catalytic domain of serum- and glucocorticoid-induced protein kinase (SGK) is 54% identical with protein kinase B (PKB) and, like PKB, is activated in vitro by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and in vivo in response to signals that activate phosphatidylinositol (PI) 3-kinase. Here we identify two novel isoforms of SGK, termed SGK2 and SGK3, whose catalytic domains share 80% amino acid sequence identity with each other and with SGK (renamed SGK1). Like SGK1, the mRNA encoding SGK3 is expressed in all tissues examined, but SGK2 mRNA is only present at significant levels in liver, kidney and pancreas and, at lower levels, in the brain. The levels of SGK2 mRNA in H4IIE cells and SGK3 mRNA in Rat2 fibroblasts are not increased by stimulation with serum or dexamethasone, whereas the level of SGK1 mRNA is increased greatly. SGK2 and SGK3 are activated in vitro by PDK1, albeit more slowly than SGK1, and their activation is accompanied by the phosphorylation of Thr193 and Thr253 respectively, the residues equivalent to the Thr in the ‘activation loop’ of PKB that is targeted by PDK1. The PDK1-catalysed phosphorylation and activation of SGK2 and SGK3, like SGK1, is greatly potentiated by mutating Ser356 and Ser419 respectively to Asp, these residues being equivalent to the C-terminal phosphorylation site of PKB. Like SGK1, SGK2 and SGK3 are activated 5-fold via a phosphorylation mechanism when cells are exposed to H2O2 but, in contrast with SGK1, activation is only suppressed partially by inhibitors of PI 3-kinase. SGK2 and SGK3 are activated to a smaller extent by insulin-like growth factor-1 (2-fold) than SGK1 (5-fold). Like PKB and SGK1, SGK2 and SGK3 preferentially phosphorylate Ser and Thr residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs.

Foreign protein antigens must be broken down within endosomes or lysosomes to generate suitable peptides that will form complexes with class II major histocompatibility complex molecules for presentation to T cells. However, it is not known which proteases are required for antigen processing. To investigate this, we exposed a domain of the microbial tetanus toxin antigen (TTCF) to disrupted lysosomes that had been purified from a human B-cell line. Here we show that the dominant processing activity is not one of the known lysosomal cathepsins, which are generally believed to be the principal enzymes involved in antigen processing, but is instead an asparagine-specific cysteine endopeptidase. This enzyme seems similar or identical to a mammalian homologue of the legumain/haemoglobinase asparaginyl endopeptidases found originally in plants and parasites. We designed competitive peptide inhibitors of B-cell asparaginyl endopeptidase (AEP) that specifically block its proteolytic activity and inhibit processing of TTCF in vitro. In vivo, these inhibitors slow TTCF presentation to T cells, whereas preprocessing of TTCF with AEP accelerates its presentation, indicating that this enzyme performs a key step in TTCF processing. We also show that N-glycosylation of asparagine residues blocks AEP action in vitro. This indicates that N-glycosylation could eliminate sites of processing by AEP in mammalian proteins, allowing preferential processing of microbial antigens.

Background: Specific inhibitors of protein kinases have great therapeutic potential, but the molecular basis underlying their specificity is only poorly understood. We have investigated the drug SB 203580 which belongs to a class of pyridinyl imidazoles that inhibits the stress-activated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38β2 but not other mitogen-activated protein kinase family members. Like inhibitors of other protein kinases, SB 203580 binds in the ATP-binding pocket of SAPK2a/p38. Results: The SAP kinases SAPK1γ/JNK1, SAPK3 and SAPK4 are not inhibited by SB 203580, because they have methionine in the position equivalent to Thr106 in the ATP-binding region of SAPK2a/p38 and SAPK2b/p38β2. Using site-directed mutagenesis of five SAP kinases and the type I and type II TGFβ receptors, we have established that for a protein kinase to be inhibited by SB 203580, the sidechain of this residue must be no larger than that of threonine. Sensitivity to inhibition by SB 203580 is greatly enhanced when the sidechain is even smaller, as in serine, alanine or glycine. Thus, the type I TGFβ receptor, which has serine at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38β2, is inhibited by SIB 203580. Conclusions: These findings explain how drugs that target the ATP-binding site can inhibit protein kinases specifically, and show that the presence of threonine or a smaller amino acid at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38β2 is diagnostic of whether a protein kinase is sensitive to the pyridinyl imidazole class of inhibitor.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).

Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6.

Purified nitrate reductase (NR) from spinach leaves was phosphorylated in vitro by NR-inactivating kinase on Ser-543 which is located in the hinge 1 region between the molybdenum-cofactor and haem-binding domains. Phosphorylation of Ser-543 allowed NR to be inhibited by the inhibitor, NIP. Degraded NR preparations in which a proportion of the subunits had lost 45 amino acids from the N-terminus during purification could be phosphorylated by NR kinase on Ser-543, but could not subsequently be fully inhibited by NIP, suggesting a role for the N-terminal tail of NR in NIP binding.

A full-length cDNA encoding the putative hepatic glycogen-binding (GL) subunit of protein phosphatase-1 (PP1) was isolated from a rat liver library. The deduced amino acid sequence (284 residues, 32.6 kDa) was 23% identical (39% similar) to the N-terminal region of the glycogen-binding (GM) subunit of PP1 from striated muscle. The similarities between GM and GL were most striking between residues 63-86, 144-166 and 186-227 of human GM (approximately 40% identity), nearly all the identities with the putative yeast homologue GAC1 being located between 144-166 and 186-227. The cDNA was expressed in E. coli, and the expressed protein transformed the properties of PP1 to those characteristic of the hepatic glycogen-associated enzyme. These experiments establish that the cloned protein is GL.

The catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase isoform 1 (HMGR1cd) from Arabidopsis thaliana has been expressed in Escherichia coli in a catalytically active form and purified. The high efficiency of the bacterial expression system together with the simplicity of the purification procedure used in this study resulted in the attainment of large quantities of pure enzyme (about 5 mg/l culture) with a final specific activity of up to 17 U/mg. This specific activity is higher than that reported to date for any 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) purified from a plant source. HMGR1cd activity was completely blocked by the HMGR inhibitor mevinolin (IC50 = 12.5 nM). No significant differences were observed between the Km values of HMGR1cd for NADPH (71 +/- 7 microM) and (S)-3-hydroxy-3-methylglutaryl-CoA (8.3 +/- 1.5 microM) and those of pure HMGR preparations obtained from different plant sources. The purified HMGR1cd was reversibly inactivated by phosphorylation at a single site by Brassica oleracea HMGR kinase A, which is functionally related to the mammalian AMP-activated protein kinase. The site of phosphorylation is Ser577 in the complete sequence of A. thaliana HMGR1. The results in this paper represent the first evidence that a higher plant HMGR is regulated by direct phosphorylation, at least in a cell-free system. Our results also reinforce the view that the AMP-activated protein kinase/SNF1 family is an ancient and highly conserved protein kinase system.

Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions. Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala. We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.

Myosin is dephosphorylated by distinct forms of protein phosphatase 1 (PP1) in smooth muscle and skeletal muscle that are composed of PP1 complexed to different regulatory subunits. The smooth muscle myosin phosphatase (smPP1M) has been characterised previously and is composed of PP1β complexed to M110 and M21 subunits that enhance the dephosphorylation of smooth muscle myosin, but not skeletal muscle myosin. In contrast, the regulatory subunit(s) of skeletal muscle myosin phosphatase (skPP1M) greatly enhance(s) the dephosphorylation of skeletal muscle myosin. Here we identify a regulatory subunit of skPP1M as the product of the MYPT2 gene, a protein whose sequence is 61% identical to the M110 subunit of smPP1M. Surprisingly, the M21 subunit of smPP1M appears to be produced from the same gene that encodes MYPT2.

A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the alpha, beta-unsaturated carbonyl of the N-methyldehydroalanine residue of MC-LR, followed by reaction of the introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound protein phosphatase-1 (PP1) with high capacity and purified human PP1 gamma in one step from E. coli extracts. It was also used to purify forms of PP1 bound to myofibrils from skeletal muscle. Two of these comprised PP1 complexed to N-terminal fragments of the M-subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N-terminal fragment of the glycogen-binding (G)-subunit.

Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited.

Background Nitrate reductase (NR) in leaves is rapidly inactivated in the dark by a two-step mechanism in which phosphorylation of NR on the serine at position 543 (Ser543) promotes binding to nitrate reductase inhibitor protein (NIP). The eukaryotic 14-3-3 proteins bind to many mammalian signalling components (Raf-1, Bcr, phosphoinositide 3-kinase, protein kinase C, polyomavirus middle-T antigen and Cdc25), and are implicated in the timing of mitosis, DNA-damage checkpoint control, exocytosis, and activation of the plant plasma-membrane H + -ATPase by fusicoccin. Their dimeric, saddle-shaped structures support the proposal that 14-3-3 proteins are ‘adaptors' linking different signalling proteins, but their precise functions are still a mystery. Results We purified NIP to homogeneity and established by means of amino-acid sequencing that it is a mixture of several 14-3-3 isoforms. Mammalian and yeast 14-3-3 proteins were just as effective as NIP at inhibiting phosphorylated NR. The sequence around Ser543, the phosphorylation site in NR, is strikingly similar to the sequences around the phosphoserine residues (Ser259 and Ser621) of mammalian Raf-1 that interact with 14-3-3 proteins. We found that NIP activity was blocked by a synthetic phosphopeptide corresponding to residues 251–266 of Raf. Fusicoccin also blocked NIP activity, and plant plasma-membrane H + -ATPases were activated by either fusicoccin, the phosphoserine259-Raf-1 peptide, or protein phosphatase 2A. Conclusions Our findings establish that the mechanism of inactivation of NR involves the phosphorylation of Ser 543 followed by interaction with one or more plant 14-3-3 proteins. These results support the idea of a common mechanism for binding of 14-3-3 to its targets in all eukaryotes, and suggest that the phosphoserine259-Raf-1 peptide and fusicoccin may be of general use for disrupting the interaction of 14-3-3 with its target proteins. We propose that the plant plasma-membrane H + -ATPase is regulated in an analogous manner to NR–NIP, and speculate that 14-3-3 proteins provide a link between ‘sensing' the activity state of NR and signalling to other cellular processes in plants.

SummaryAutophosphorylation of a critical residue in the activation loop of several protein kinases is an essential maturation event required for full enzyme activity. However, the molecular mechanism by which this happens is unknown. We addressed this question for two dual-specificity tyrosine-phosphorylation-regulated protein kinases (DYRKs), as they autophosphorylate their activation loop on an essential tyrosine but phosphorylate their substrates on serine and threonine. Here we demonstrate that autophosphorylation of the critical activation-loop tyrosine is intramolecular and mediated by the nascent kinase passing through a transitory intermediate form. This DYRK intermediate differs in residue and substrate specificity, as well as sensitivity to small-molecule inhibitors, compared with its mature counterpart. The intermediate’s characteristics are lost upon completion of translation, making the critical tyrosine autophosphorylation a “one-off” inceptive event. This mechanism is likely to be shared with other kinases.

Clathrin light chain (CL) b purified from bovine brain postmicrotubule supernatant and identified by mass spectrometry potently inhibited a catalytic activity of a major protein phosphatase (PP) that was copurified with microtubules and recognized by antiPP1 antibodies. CLb similarly affected the catalytic subunit and holoenzyme of the PP, little inhibiting the activity of PP2A. Although the CLb from clathrin-coated vesicles was several hundredfold weaker than our purified CLb, the CLb in the postmicrotubule supernatant, independent of whether it was sedimentable or soluble, was as active as the purified CLb. Thus CLb may be a potent regulator of the PP.

Proteins of approximately 35, 55 and 65kDa were purified from cauliflower extracts by microcystin-Sepharose chromatography and identified by amino acid sequencing as plant forms of protein (serine/threonine) phosphatase 1 (PP1) catalytic subunit, PP5 and a regulatory A-subunit of PP2A, respectively. Peptides that corresponded both to the tetratricopeptide (TPR) repeat and catalytic domains of PP5 were identified. Similar to mammalian PP5,the casein phosphatase activity of plant PP5 was activated10-fold by arachidonic acid, with half-maximal stimulation occurring at approximately 100 microM lipid.