Search

Although the physiological roles of p38γ and p38δ signalling pathways are largely unknown, new genetic and pharmacological tools are providing groundbreaking information on the function of these two stress-activated protein kinases. Recent studies show the importance of p38γ and p38δ in the regulation of processes as diverse as cytokine production, protein synthesis, exocytosis, cell migration, gene expression, and neuron activity, which have an acute impact on the development of pathologies related to inflammation, diabetes, neurodegeneration, and cancer. These recent breakthroughs are resolving some of the questions that have long been asked regarding the function of p38γ and p38δ in biology and pathology.

© 2021 by the authors., p38 Mitogen activated protein kinases (p38MAPK) are a highly evolutionary conserved group of protein kinases, which are central for cell adaptation to environmental changes as well as for immune response, inflammation, tissue regeneration, and tumour formation [...], This research was funded by grants from the Spanish Ministerio of Ciencia e Innovación (MICINN) (SAF2016-79792R, PID2019-108349RB-I00).

Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of μ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor μ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. J. Cell. Biochem. 117: 2781-2790, 2016. © 2016 Wiley Periodicals, Inc.

Non-alcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen-activated kinases (p38γ and p38δ) have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38δ is elevated in livers of obese patients with NAFLD and that mice lacking p38γ/δ in myeloid cells are resistant to diet-induced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38γ/δ-deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild-type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38γ and p38δ in myeloid cells provide a potential target for NAFLD therapy.

p38 MAPKs regulate migration and invasion. However, the mechanisms involved are only partially known. We had previously identified fibulin 3, which plays a role in migration, invasion, and tumorigenesis, as a gene regulated by p38α. We have characterized in detail how p38 MAPK regulates fibulin 3 expression and its role. We describe here for the first time that p38α, p38γ, and p38δ down-regulate fibulin 3 expression. p38α has a stronger effect, and it does so through hypermethylation of CpG sites in the regulatory sequences of the gene. This would be mediated by the DNA methylase, DNMT3A, which is down-regulated in cells lacking p38α, but once re-introduced represses Fibulin 3 expression. p38α through HuR stabilizes dnmt3a mRNA leading to an increase in DNMT3A protein levels. Moreover, by knocking-down fibulin 3, we have found that Fibulin 3 inhibits migration and invasion in MEFs by mechanisms involving p38α/β inhibition. Hence, p38α pro-migratory/invasive effect might be, at least in part, mediated by fibulin 3 down-regulation in MEFs. In contrast, in HCT116 cells, Fibulin 3 promotes migration and invasion through a mechanism dependent on p38α and/or p38β activation. Furthermore, Fibulin 3 promotes in vitro and in vivo tumor growth of HCT116 cells through a mechanism dependent on p38α, which surprisingly acts as a potent inducer of tumor growth. At the same time, p38α limits fibulin 3 expression, which might represent a negative feed-back loop.

Objective The role of most p38 MAPK isoforms in inflammatory arthritis is not known. This study was undertaken to evaluate p38γ and p38δ deficiency in the collagen-induced arthritis (CIA) model. Methods Wild-type, p38γ−/−, p38δ−/−, and p38γ/δ−/− mice were immunized with chicken type II collagen, and disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum cytokine levels and in vitro T cell cytokine responses were quantified by flow cytometry and multiplex analysis, and serum anticollagen antibody levels by enzyme-linked immunosorbent assay. Cytokine and p38 MAPK isoform expression in joints were determined by quantitative polymerase chain reaction. Results Compound p38γ and p38δ deficiency markedly reduced arthritis severity compared with that in wild-type mice, whereas lack of either p38γ or p38δ had an intermediate effect. Joint damage was minimal in arthritic p38γ/δ−/− mice compared with wild-type mice. The p38γ/δ−/− mice had lower levels of pathogenic anticollagen antibodies and interleukin-1β (IL-1β) and tumor necrosis factor α than controls. In vitro T cell assays showed reduced proliferation, interferon-γ (IFNγ) production, and IL-17 production by lymph node cells from p38γ/δ−/− mice. IL-17 and IFNγ messenger RNA expression in joints was significantly inhibited in p38γ/δ−/− mice. Wild-type chimeric mice with p38γ/δ−/− bone marrow did not show decreased CIA. Conclusion Reduced disease severity in p38γ/δ−/− mice was associated with lower cytokine production and anticollagen antibody responses than in controls, indicating that p38γ and p38δ are crucial regulators of inflammatory joint destruction in CIA. Our findings indicate that p38γ and p38δ are potential therapeutic targets in complex diseases, such as rheumatoid arthritis, that involve innate and adaptive immune responses.

Dendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)-directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK-regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.

Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

The regenerative capacity of muscle is regulated by p38-γ, which phosphorylates MyoD and leads to formation of a complex that represses myogenin transcription., The mitogen-activated protein kinase p38-γ is highly expressed in skeletal muscle and is associated with the dystrophin glycoprotein complex; however, its function remains unclear. After induced damage, muscle in mice lacking p38-γ generated significantly fewer myofibers than wild-type muscle. Notably, p38-γ-deficient muscle contained 50% fewer satellite cells that exhibited premature Myogenin expression and markedly reduced proliferation. We determined that p38-γ directly phosphorylated MyoD on Ser199 and Ser200, which results in enhanced occupancy of MyoD on the promoter of myogenin together with markedly decreased transcriptional activity. This repression is associated with extensive methylation of histone H3K9 together with recruitment of the KMT1A methyltransferase to the myogenin promoter. Notably, a MyoD S199A/S200A mutant exhibits markedly reduced binding to KMT1A. Therefore, p38-γ signaling directly induces the assembly of a repressive MyoD transcriptional complex. Together, these results establish a hitherto unappreciated and essential role for p38-γ signaling in positively regulating the expansion of transient amplifying myogenic precursor cells during muscle growth and regeneration.

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is a component of the p38γ MAP kinase pathway, which is important for the adaptation of mammalian cells to changes in environmental osmolarity. Here we report a strong decrease in the levels of hDlg protein in the human epithelial cell line HeLa when exposed to osmotic shock. This is independent of the phosphorylation state of hDlg, is prevented by preincubating the cell with the caspase inhibitor z-VAD and is part of the apoptotic process triggered by cellular stress. Although, both caspase 3 and caspase 6 are strongly activated by osmotic shock, the time course of caspase 6 activation parallels hDlg degradation, suggesting that this caspase may be responsible for the proteolysis. Mutating hDlg Asp747 to Ala abolishes caspase-induced cleavage, but does not affect the early stage of apoptosis or cell attachment. Our findings show that osmotic stress triggers hDlg degradation through a mechanism different from the one mediated by proteasomes, and we identify hDlg as a caspase substrate during the apoptotic process, although its proteolysis may not be implicated in the progression of early apoptosis.

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.

Mammalian p38 mitogen-activated protein kinases (MAPKs) are activated by a wide range of cellular stresses as well as in response to inflammatory cytokines. There are four members of the p38MAPK family (p38alpha, p38beta, p38gamma and p38delta) which are about 60% identical in their amino acid sequence but differ in their expression patterns, substrate specificities and sensitivities to chemical inhibitors such as SB203580. A large body of evidences indicates that p38MAPK activity is critical for normal immune and inflammatory response. The p38MAPK pathway is a key regulator of pro-inflammatory cytokines biosynthesis at the transcriptional and translational levels, which makes different components of this pathway potential targets for the treatment of autoimmune and inflammatory diseases. However, recent studies have shed light on the broad effect of p38MAPK activation in the control of many other aspects of the physiology of the cell, such as control of cell cycle or cytoskeleton remodelling. Here we focus on these emergent roles of p38MAPKs and their implication in different pathologies.

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.

Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38gamma MAP kinase (SAPK3/p38gamma) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38gamma-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular-junctional complexes, and cell shape and volume in response to osmotic stress.

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK IC 50 = 3.0 ± 0.1 μM , n = 3 and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%–70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase IC 50 = 3.3 ± 1.4 μM , n = 3 and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50–90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.

Mechanisms for selective targeting to unique subcellular sites play an important role in determining the substrate specificities of protein kinases. Here we show that stress-activated protein kinase-3 (SAPK3, also called ERK6 and p38γ), a member of the mitogen-activated protein kinase family that is abundantly expressed in skeletal muscle, binds through its carboxyl-terminal sequence -KETXL to the PDZ domain of α1-syntrophin. SAPK3 phosphorylates α1-syntrophin at serine residues 193 and 201 in vitro and phosphorylation is dependent on binding to the PDZ domain of α1-syntrophin. In skeletal muscle SAPK3 and α1-syntrophin co-localize at the neuromuscular junction, and both proteins can be co-immunoprecipitated from transfected COS cell lysates. Phosphorylation of a PDZ domain-containing protein by an associated protein kinase is a novel mechanism for determining both the localization and the substrate specificity of a protein kinase.

PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and a mutant MAPKK1(S217E,S221E), which has low levels of constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC50 = 2-7 microM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 microM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates, indicating that it exerts its effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in Swiss 3T3 cells, suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059 in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in KB and PC12 cells, and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD 098059 (50 microM) inhibited the activation of p42MAPK and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells, but the extent of inhibition depended on how potently c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor, serum, insulin, and phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth factor was increased 3-fold, and the subsequent inactivation that occurred after 10 min was prevented. These results indicate that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP kinase pathway.

The uptake of 2-deoxyglucose into KB cells was stimulated about 2-fold by interleukin-1 (IL1), anisomycin or insulin-like growth factor-1 (IGF1). Stimulation by IL1 and anisomycin was prevented by SB 203580, a specific inhibitor of the mitogen-activated protein (MAP) kinase homologue termed ‘re-activating kinase’ [RK; also known as p38, p40 and CSBP (cytokine synthesis anti-inflammatory-drug-binding protein)], but was unaffected by PD 98059, a specific inhibitor of the activation of the classical MAP kinase pathway. In contrast, the stimulation of 2-deoxyglucose uptake by IGF1 was blocked by PD 98059 and unaffected by SB 203580. Consistent with these observations, IL1 and anisomycin were potent activators of MAP kinase-activated protein (MAPKAP) kinase-2, a physiological substrate of RK, whereas IGF1 was only a very weak activator of MAPKAP kinase-2. Conversely, IGF1 was a stronger activator of p42 MAP kinase than IL1 or anisomycin. These results imply that the activation of distinct MAP kinase pathways is required for the stimulation of glucose transport by IL1/anisomycin and IGF1 in KB cells, and suggest that the combined use of SB 203580 and PD 98059 is a powerful new approach to explore the roles of different MAP kinase cascades in cell regulation.

Skeletal muscle glycogen phosphorylase b binds to sarcoplasmic reticulum (SR) membranes with a dissociation constant of 1.7 +/- 0.6 mg of phosphorylase/ml at 25 degrees C at physiological pH and ionic strength. Raising the temperature to 37 degrees C produced a 2-3-fold decrease in the dissociation constant. The SR membranes could bind up to 1.1 +/- 0.1 mg of glycogen phosphorylase b/mg of SR protein, whereas liposomes prepared with endogenous SR lipids and reconstituted Ca(2+)-ATPase were unable to bind glycogen phosphorylase. Binding of glycogen phosphorylase b to SR membranes is accompanied by inhibition of its activity in the presence of AMP. The Vmax for glycogen phosphorylase b associated with SR membranes is 40 +/- 5% of that for purified glycogen phosphorylase and shows a decreased affinity for its allosteric activators, AMP and IMP. These kinetic effects are also observed with purified glycogen phosphorylase b when starch or alpha-amylose is used as substrate instead of glycogen. Treatment of SR membranes with alpha-amylase produced dissociation of glycogen phosphorylase b from the SR membranes. Thus, linear polysaccharide fragments of glycogen bound to the SR membranes are likely mediating the binding of glycogen phosphorylase b to these membranes.

Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38gamma MAPK, and the activated c-Jun then recruits p38gamma as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38gamma that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38gamma interacting with c-Jun. p38gamma requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38gamma are required for the trans-activation of MMP9. The active p38gamma/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38gamma and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response.

All four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family (p38alpha, p38beta, p38gamma and p38delta) are activated by dual phosphorylation in the TGY motif in the activation loop. This phosphorylation is mediated by three kinases, MKK3, MKK6 and MKK4, at least in vitro. The role of these MKK in the activation of p38alpha has been demonstrated in studies using fibroblasts that lack MKK3 and/or MKK6. Nonetheless, the physiological upstream activators of the other p38MAPK isoforms have not yet been reported using MKK knockout cells. In this study, we examined p38beta, gamma and delta activation by MKK3 and MKK6, in cells lacking MKK3, MKK6 or both. We show that MKK3 and MKK6 are both essential for the activation of p38gamma and p38beta induced by environmental stress, whereas MKK6 is the major p38gamma activator in response to TNFalpha. In contrast, p38delta activation by ultraviolet radiation, hyperosmotic shock, anisomycin or by TNFalpha is mediated by MKK3. Moreover, in response to osmotic stress, MKK3 and MKK6 are crucial in regulating the phosphorylation of the p38gamma substrate hDlg and its activity as scaffold protein. These data indicate that activation of distinct p38MAPK isoforms is regulated by the selective and synchronized action of two kinases, MKK3 and MKK6, in response to cell stress.

The Discs Large (Dlg) protein is intimately involved in regulating cell polarity and cell proliferation, and is targeted by the high-risk Human Papillomavirus (HPV) E6 proteins for proteasome-mediated degradation. We show here that exposure of cells to osmotic shock induces the hyperphosphorylation of Dlg and its concomitant accumulation within the cell membrane at sites of cell contact, a process that requires an intact actin filament network. In addition, hyperphosphorylation of Dlg also renders it more susceptible to degradation induced by the HPV-18 E6 oncoprotein. Mutational analysis of Dlg identifies a region within the first 185 amino acids as being important for this, with phosphorylation on residue S158 being responsible for its enhanced targeting by the E6 oncoprotein. Using specific kinase inhibitors, we show that Jun N-terminal kinase (JNK) is in part responsible for this phosphorylation, and for the subsequent Dlg accumulation at sites of cell contact. These results further support the notion of a complex phosphorylation-dependent regulation of Dlg, both with respect to its precise cellular localisation and to its susceptibility to proteasome-mediated degradation.

The compound BIRB796 inhibits the stress-activated protein kinases p38alpha and p38beta and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38gamma. This occurs at higher concentrations of BIRB796 than those that inhibit p38alpha and p38beta and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38gamma. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38alpha and p38beta, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38gamma as well as those of p38alpha and p38beta.

SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli.

The endogenous glycogen synthase in extracts from mouse skeletal muscle, liver and brain bound specifically to SAPK2b (stress-activated protein kinase 2b)/p38β, but not to other members of the group of SAPK/p38 kinases. Glycogen synthase was phosphorylated in vitro more efficiently by SAPK2b/p38β than by SAPK2a/p38α, SAPK3/p38γ or SAPK4/p38δ. SAPK2b/p38β phosphorylated glycogen synthase in vitro at residues Ser644, Ser652, Thr718 and Ser724, two of which (Ser644 and Ser652) are also phosphorylated by glycogen synthase kinase 3. Thr718 and Ser724 are novel sites not known to be phosphorylated by other protein kinases. Glycogen synthase becomes phosphorylated at Ser644 in response to osmotic shock; this phosphorylation is prevented by pretreatment of the cells with SB 203580, which inhibits SAPK2a/p38α and SAPK2b/p38β activity. In vitro, phosphorylation of glycogen synthase by SAPK2b/p38β alone had no significant effect on its activity, indicating that phosphorylation at residue Ser644 itself is insufficient to decrease glycogen synthase activity. However, after phosphorylation by SAPK2b/p38β, subsequent phosphorylation at Ser640 by glycogen synthase kinase 3 decreased the activity of glycogen synthase. This decrease was not observed when SAPK2b/p38β activity was blocked with SB 203580. These results suggest that SAPK2b/p38β may be a priming kinase that allows glycogen synthase kinase 3 to phosphorylate Ser640 and thereby inhibit glycogen synthase activity.

The biochemical mechanism of apoptosis induced by ceramide remains still unclear, although it has been reported that dephosphorylation of PKB at Ser-473 may be a key event. In this article, we show that C(2)-ceramide (N-acetyl-sphingosine) induces the dephosphorylation of both protein kinase B (PKB) and glycogen synthase kinase-3 (GSK3) in cerebellar granule cells (CGC). We also show that lithium protects against the apoptosis induced by C(2)-ceramide by blocking the dephosphorylation of both kinases. Since lithium inhibits in vivo the observed protein phosphatase-2A (PP2A) activation induced by ceramide, we hypothesise that the neuroprotective action of lithium may be due to the inhibition of the PP2A activation by apoptotic stimuli.

The differentiation of C2C12 myoblasts to myotubes was found to be accompanied by a strong activation of p70 S6 kinase and the mitogen-activated protein kinase (MAPK) family member SAPK2/p38, without significant activation of p42 MAPK and only slight activation of SAPK1/JNK and protein kinase Balpha. Consistent with these findings, SB 203580 (a specific inhibitor of SAPK2/p38) or rapamycin (which blocks the activation of p70 S6 kinase) prevented the formation of multinucleated myotubes, as well as the expression of muscle-specific proteins that included SAPK3 (another MAPK family member). PD 098059 (which prevents the activation of p42 MAPK) had no effect on myotube formation. Surprisingly, the slow activation of p70 S6 kinase during differentiation was not only prevented by rapamycin but also by SB 203580, and the activation of MAPKAP kinase-2 (an in vivo substrate of SAPK2/p38) was not only prevented by SB 203580 but also by rapamycin. In contrast, the acute activation of p70 S6 kinase in C2C12 myoblasts induced by phorbol esters was unaffected by SB 203580 and the acute activation of MAPKAP kinase-2 induced by anisomycin was unaffected by rapamycin. These results show for the first time that SAPK2/p38 plays an essential role in C2C12 cell differentiation.

Overexpression of the protein kinases mixed-lineage kinase-2 (MLK2) or mitogen-activated protein kinase (MAPK) kinase kinase-1 (MEKK1) is known to trigger the activation of stress-activated protein kinase (SAPK1)/c-Jun N-terminal kinase (JNK). Here we demonstrate that MLK2 activates SAPK kinase-1 (SKK1)/MAPK kinase 4 (MKK4) and SKK4/MKK7, the two known direct activators of SAPK1/JNK (both in transfection studies and in vitro). In contrast, MEKK1 activates SKK1/MKK4 more efficiently than MLK2, but barely activates SKK4/MKK7. Since SKK4/MKK7 (but not SKK1/MKK4) is activated by interleukin-1 and tumour necrosis factor in several cells and tissues, we suggest that MEKK1 does not mediate the activation of SKK4/MKK7 and SAPK1/JNK induced by these pro-inflammatory cytokines. MLK2 and MEKK1 also activated SKK2/MKK3 and SKK3/MKK6, the direct upstream activators of SAPK2a/p38.

The ERK, JNK/SAPK and p38/RK MAP kinase subtypes are differentially activated by physiological, pharmacological and stress stimuli; all three subtypes are implicated in immediate-early (IE) gene induction by these agents. Here, we have asked whether inhibition of a single MAP kinase subtype under these conditions would generally alter induction of several IE genes in a similar way or whether this would differentially up- and down-regulate particular IE genes, an issue which bears on the question of whether individual MAP kinases are strictly targeted to specific IE genes, or whether they might catalyse phosphorylation events that affect several IE genes in the same way. SB 203580, an inhibitor of p38/RK, has been used to analyse the role of this kinase in the induction of five IE genes (c-fos, fosB, c-jun, junB and junD) under diverse conditions of stimulation. In C3H 10T1/2 cells, p38/RK and its downstream kinase MAPKAP K-2 are activated by all stimuli used with the exception of TPA. The specificity of SB 203580 as a p38/RK inhibitor in these cells is demonstrated; it does not affect ERKs or JNK/SAPKs but does result in a small increase in the activity of the upstream kinase MKK6, the principal p38/RK activator in these cells. We find that inhibition of p38/RK under these conditions produces general effects on all five IE genes as a group in three ways. First, induction of all five genes in response to okadaic acid or tumour necrosis factor-alpha (TNF-alpha) is not significantly altered by SB 203580. Second, in cells stimulated with anisomycin or U.V. radiation, SB 203580 potently inhibits all of the induced IE genes. Finally, SB 203580 enhances induction of all five IE genes in EGF-treated cells; these enhanced mRNA levels are not due to stabilisation of labile mRNA transcripts. The significance of these results to current thinking on the relationship between distinct MAP kinase subtypes and specific IE genes is discussed.

A cDNA was cloned and expressed that encodes human stress-activated protein kinase kinase-4 (SKK4), a novel MAP kinase kinase family member whose mRNA is widely expressed in human tissues. SKK4 activated SAPK1/JNK in vitro, but not SAPK2a/p38, SAPK2b/p38β, SAPK3/ERK6 or SAPK4. It appears to be the mammalian homologue of HEP, an activator of SAPK1/JNK in Drosophila. In human epithelial KB cells SKK4 and SKK1/MKK4 (another activator of SAPK1/JNK) were both activated by stressful stimuli, but only SKK4 was activated by proinflammatory cytokines. The identification of SKK4 explains why the major SAPK1/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4.

A cDNA was cloned that encodes human stress-activated protein kinase-4 (SAPK4), a novel MAP kinase family member whose amino acid sequence is approximately 60% identical to that of the other three SAP kinases which contain a TGY motif in their activation domain. The mRNA encoding SAPK4 was found to be widely distributed in human tissues. When expressed in KB cells, SAPK4 was activated in response to cellular stresses and pro-inflammatory cytokines, in a manner similar to other SAPKs. SAPK4 was activated in vitro by SKK3 (also called MKK6) or when co-transfected with SKK3 into COS cells. SKK3 was the only activator of SAPK4 that was induced when KB cells were exposed to a cellular stress or stimulated with interleukin-1. These findings indicate that SKK3 mediates the activation of SAPK4. The substrate specificity of SAPK4 in vitro was similar to that of SAPK3. Both enzymes phosphorylated the transcription factors ATF2, Elk-1 and SAP-1 at similar rates, but were far less effective than SAPK2a (also called RK/p38) or SAPK2b (also called p38beta) in activating MAPKAP kinase-2 and MAPKAP kinase-3. Unlike SAPK1 (also called JNK), SAPK3 and SAPK4 did not phosphorylate the activation domain of c-Jun. Unlike SAPK2a and SAPK2b, SAPK4 and SAPK3 were not inhibited by the drugs SB 203580 and SB 202190. Our results suggest that cellular functions previously attributed to SAPK1 and/or SAPK2 may be mediated by SAPK3 or SAPK4.

Stress-activated protein kinase-3 (SAPK3), a recently described MAP kinase family member with a wide-spread tissue distribution, was transfected into several mammalian cell lines and shown to be activated in response to cellular stresses, interleukin-1 (IL-1) and tumour necrosis factor (TNF) in a similar manner to SAPK1 (also termed JNK) and SAPK2 (also termed p38, RK, CSBP and Mxi2). SAPK3 and SAPK2 were activated at similar rates in vitro by SAPKK3 (also termed MKK6), and SAPKK3 was the only activator of SAPK3 that was induced when KB or 293 cells were exposed to cellular stresses or stimulated with IL-1 or TNF. Co-transfection with SAPKK3 induced SAPK3 activity and greatly enhanced activation in response to osmotic shock. These experiments indicate that SAPKK3 mediates the activation of SAPK3 in several mammalian cells. SAPK3 and SAPK2 phosphorylated a number of proteins at similar rates, including the transcription factors ATF2, Elk-1 and SAP1, but SAPK3 was far less effective than SAPK2 in activating MAPKAP kinase-2 and MAPKAP kinase-3. Unlike SAPK2, SAPK3 was not inhibited by the drug SB 203580. SAPK3 phosphorylated ATF2 at Thr69, Thr71 and Ser90, the same residues phosphorylated by SAPK1, whereas SAPK2 only phosphorylated Thr69 and Thr71. Our results suggest that cellular functions previously attributed to SAPK1 and/or SAPK2 may be mediated by SAPK3.

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.

The ERK, JNK/SAPK and p38/RK MAP kinase subtypes (reviewed in [1]) are differentially activated in mammalian cells by various stimuli, which elicit induction of immediate-early (IE) genes, such as c-fos and c-jun (reviewed in [1-3]), as well as phosphorylation of histone H3 [4] and HMG-14 [5]. Anisomycin and UV radiation have been suggested to induce c-fos and c-jun transcription via JNK/SAPK-mediated phosphorylation of TCF (ternary complex factor), for c-fos induction [6-8], and c-Jun and/or ATF-2 for c-jun induction [9-11] [12,13]. We report here that anisomycin and ultraviolet radiation (UV) activate MAP kinase kinase-6 (MKK6) [14,15], p38/RK [16] [17,18] and MAPKAP kinase-2 (MAPKAP K-2) [17-19]. By using the p38/RK inhibitor SB 203580 [20,21], we show that activation of p38/RK and/or its downstream effectors are essential for anisomycin- and UV-stimulated c-fos/c-jun induction and histone H3/HMG-14 phosphorylation, whereas JNK/SAPK activation and phosphorylation of c-Jun and ATF-2 are insufficient for these responses.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.

The identities of the upstream activators of the mitogen-activated protein (MAP) kinase homologues termed stress-activated-protein (SAP) kinase-1 (also known as JNK or SAPK) and SAP kinase-2 (also known as p38, RK and CSBP) were investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators, SAP kinase kinase-1 (SAPKK-1) and SAPKK-2 were activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin, and more weakly in response to sodium arsenite. SAPKK-1 was capable of activating both SAP kinase-1 and SAP kinase-2 and was similar, if not identical, to the previously described MAP kinase kinase homologue MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK-2 activated SAP kinase-2, but not SAP kinase-1 in vitro. In KB cells, five distinct upstream activators of SAP kinase-1 and SAP kinase-2 were induced, namely SAPKK-1, SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, whose appearance depended on the nature of the stimulus. SAPKK-3, which was strongly induced by every stimulus tested (osmotic shock, ultraviolet irradiation, anisomycin or IL-1), accounted for about 95% of the SAP kinase-2 activator activity in these cells, did not activate SAP kinase-1 and eluted from Mono S at a lower salt concentration than SAPKK-2. SAPKK-4 and SAPKK-5 were also eluted from Mono S at higher NaC1 concentrations than SAPKK-3 and these enzymes activated SAP kinase-1 but not SAP kinase-2. SAPKK-4 was the only SAP kinase-1 activator induced by interleukin-1 or ultraviolet irradiation, while two SAP kinase-1 activators, SAPKK-1 and SAPKK-5, were induced by osmotic shock or anisomycin. SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, were not activated by MEK kinase in vitro, were separable from the major activator(s) of p42 MAP kinase, and were not recognised by anti-MKK4 antibodies. At least two of these enzymes are likely to be novel MAP kinase kinase homologues. Our results demonstrate unexpected complexity in the upstream regulation of stress and cytokine-stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type.

A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739–746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC50 = 0.6 μM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.

In the presence of glycogen, ADP, phosphoglucomutase and hexokinase, the glycogen phosphorylase b activity associated to sarcoplasmic reticulum (SR) membranes stimulates Ca2+ uptake by SR membrane fragments in the absence of added ATP. Phosphoglucomutase and hexokinase lead to the formation of glucose 6-phosphate which in turn is used as an ATP regenerating system by the Ca2+ pump. It is proposed that a raise of cytosolic AMP and ADP concentrations after muscle contraction can activate an alternative metabolic route which would be used to ensure the maintenance of a low cytosolic Ca2+ concentration and avoid unnecessary metabolic energy depletion in muscle cells.

Glycosen phosphorylase b at concentrations close to those found in skeletal muscle interacts with sarcoplasmic reticulum membranes, but not with liposomes made of lipids extracted from these membranes, and is inhibited upon binding to the membrane. The interaction of glycogen phosphorylase with the sarcoplasmic reticulum membrane is modulated by phosphorylation, for the a form of this enzyme shows a K0.5 of interaction about 10-fold lower than the b form. Upon association to the membrane the fluorescence properties of the coenzymes of glycogen phosphorylase, pyridoxal-5′-phosphate, are strongly altered, for the fluorescence at 535 nm is partially quenched and the fluorescence at 415–420 nm increases. Using fluorescein labeled sarcoplasmic reticulum membranes we have found that average conformation of the Ca2+ + Mg2+-ATPaze is also altered on binding or phosphorylase b. In conclusion, the results reported in this paper suggest that glycogen phosphorylase and Ca2+ + Mg2+-ATPase directly interact under experimental conditions similar to those found in the sarcoplasm, and that this interaction is modulated by phosphorylation of the phosphorylase.

The paired helical filament, which comprises the major fibrous element of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated microtubule-associated protein tau. Many of the hyperphosphorylated sites in tau are serine/threonine-prolines. Here we show that the stress-activated protein (SAP) kinases SAPK1gamma (also called JNK1), SAPK2a (also called p38, RK, CSBPs, Mpk2 and Mxi2), SAPK2b (also called p38beta), SAPK3 (also called ERK6 and p38gamma) and SAPK4 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Based on initial rates of phosphorylation, tau was found to be a good substrate for SAPK4 and SAPK3, a reasonable substrate for SAPK2b and a relatively poor substrate for SAPK2a and SAPK1gamma. Phosphorylation of tau by SAPK3 and SAPK4 resulted in a marked reduction in its ability to promote microtubule assembly. These findings double the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.

A MAP kinase homologue, termed the reactivating kinase (RK), lies in a signalling pathway which mediates cellular responses to stress. Here we demonstrate that the stress-induced activation of the RK in human KB cells is accompanied by the phosphorylation of Thr-180 and Tyr-182, and that the phosphorylation of both residues is required for the activation of this enzyme.