Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization

While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M _r 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca²⁺ channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M _r 32 000 required protein phosphatase 1/2A activity and extracellular Ca²⁺. In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na⁺ or Ca²⁺ sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.     

Differential regulation of M3/6 (DUSP8) signaling complexes in response to arsenite-induced oxidative stress

Mitogen-activated protein kinase (MAPK) cascades are involved in the regulation of cellular proliferation, differentiation, survival, apoptosis, as well as in inflammatory responses. Signal intensity and duration have been recognized as crucial parameters determining MAPK signaling output. Phosphatases play a particularly important role in this respect, by tightly controlling MAPK phosphorylation and activation. M3/6 (DUSP8) is a dual-specificity phosphatase implicated in the dephosphorylation and inactivation of JNK and, to a lesser extent, p38 MAPKs and is found in a complex with these kinases, along with other pathway components, held together by scaffold proteins. The JNK family consists of three genes, giving rise to at least ten different splice variants. Some functional differences between these gene products have been demonstrated, but the underlying molecular mechanisms and the roles of individual splice variants are still incompletely understood. We have investigated the interaction of M3/6 with JNK isoforms, as well as scaffold proteins of the JNK interacting protein (JIP) family, in order to elucidate the contribution of M3/6 to the regulation of distinct JNK signaling modules. M3/6 exhibited stronger binding towards JNK1β and JNK2α isoforms and this was reflected in higher enzymatic activity towards JNK2α2 when compared to JNK1α1 in vitro. After activation of the pathway by exposure of cells to arsenite, the interaction of M3/6 with JNK1α and JNK3 was enhanced, whereas that with JNK1β or JNK2α decreased. The modulation of binding affinities was found to be independent of JNK-mediated M3/6 phosphorylation. Furthermore, arsenite treatment resulted in an inducible recruitment of M3/6 to JNK-interacting protein 3 (JIP3) scaffold complexes, while its interaction with JIP1 or JIP2 was constitutive. The presented data suggest an isoform-specific role for the M3/6 phosphatase and the dynamic targeting of M3/6 towards distinct JNK-containing signaling complexes.     

Identification of a novel Ser/Thr protein phosphatase Ppq1 as a negative regulator of mating MAP kinase pathway in Saccharomyces cerevisiae

The specificity and efficiency of cell signaling is largely governed by the complex formation of signaling proteins. The precise spatio-temporal control of the complex assembly is crucial for proper signaling and cell survival. Protein phosphorylation is a key mechanism of signal processing in most of cell signaling networks. Phosphatases, along with kinases, control the phosphorylation state of many proteins and thus play a critical role in the precise regulation of signaling at each stage such as activation, propagation, and adaptation. Identification and functional analysis of pathway-specific phosphatase is, therefore, crucial for the understanding of cell signaling mechanisms. Here, we have developed a novel screening strategy to identify pathway-specific phosphatases, in which the entire repertoire of cell’s phosphatases was tethered to a signaling complex and the changes in signaling response were monitored. As a model target, we have chosen the mating MAP kinase pathway in the budding yeast, which is composed of three kinases and Ste5 scaffold protein. Using this strategy, a putative Ser/Thr phosphatase, Ppq1, was identified to be mating-specific. Results show that Ppq1 down-regulates mating signaling by targeting at or upstream of the terminal MAP kinase Fus3 in the cascade. The catalytic activity of Ppq1 as a phosphatase was confirmed in vitro and is necessary for its function in the regulation of mating signaling. Overall, the data suggest that Ppq1 functions as a negative regulator of mating MAPK pathway by dephosphorylating target pathway protein(s) and plays a key role in the control of the background signaling noise.     

The Drosophila DUSP puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress

Dual-Specificity Phosphatases (DUSPs) are enzymes that remove phosphate groups from both phospho-tyrosine and phospho-serine/threonine residues. A subgroup of DUSPs specifically targets Mitogen-Activated Protein Kinases (MAPKs) and has been shown to participate in the regulation of differential cellular responses to the large variety of stimuli conveyed by MAPK-pathways. In Drosophila, Puckered has been identified as a DUSP, exhibiting specificity towards the c-Jun-N-terminal kinase (JNK). Recent studies have signified its role in regulating JNK-dependent processes, including immunity, stress tolerance and longevity. Puckered expression depends on the activation of the JNK pathway whereas it's degradation is mediated by the ubiquitin-proteasome system. In this study we show that Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress and that phosphorylation affects the interaction between Puckered and these MAPKs. In silico analysis of the Puckered amino acid sequence revealed several MAPK consensus phosphorylation motifs. Expression of Puckered in the heterologous system of HEK293 cells and subsequent stimulation with arsenite resulted in reduced mobility of Puckered in SDS-PAGE. Similar results were obtained when Puckered was co-expressed with the constitutively active forms of JNK and p38. This mobility shift was abolished by lambda-phosphatase treatment or by simultaneous inhibition of JNK and p38. Analysis by mass-spectrometry identified Puckered phosphorylation in Ser413, though phosphorylation on this site was found irrespective of stimulation. Finally, phosphorylation of Puckered enhanced its interaction both with JNK and p38. Our results suggest a possible functional role of Puckered phosphorylation by MAPKs.     

The JNK phosphatase M3/6 is inhibited by protein-damaging stress

Cells respond to stresses such as osmotic shock and heat shock by activating stress-activated protein kinases (SAPKs), including c-Jun N-terminal kinase (JNK) [1]. Activation of JNK requires phosphorylation of threonine and tyrosine residues in the TPY activation loop motif [2, 3] and can be reversed by the removal of either phosphate group. Numerous JNK phosphatases including dual-specificity phosphatases [4, 5], have been identified. Many stimuli activate JNK by increasing its rate of phosphorylation; however, JNK dephosphorylation is inhibited in cells after heat shock [6], suggesting that a JNK phosphatase(s) is inactivated. M3/6 is a dual-specificity phosphatase selective for JNK [7, 8]. We have previously expressed M3/6 in the mouse bone marrow cell line BAF3 in order to show that JNK activation by IL-3 is necessary for cell survival and proliferation [9]. Here we report that M3/6 dissociates from JNK and appears in an insoluble fraction after heat shock. These data identify M3/6 as a JNK phosphatase that is inactivated by heat shock and provide a molecular mechanism for the activation of JNK by heat shock.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.     

Dephosphorylation of the human T lymphocyte CD3 antigen

Previous studies demonstrated that activation of T lymphocytes by phorbol ester or mitogenic lectin leads to phosphorylation of Ser 126 of the CD3 antigen gamma chain, whereas treatment with ionomycin results in phosphorylation of both Ser 123 and 126 [Davies, A. A. et al. (1987) J. Biol. Chem. 262, 10918-10921]. In the present study, the dephosphorylation of Ser 123 and Ser 126 of the gamma chain was investigated. Phorbol-ester-induced phosphorylation of the gamma-chain Ser 126 in vivo was reversed following removal of phorbol ester. Dephosphorylation of both Ser 123 and 126 was also observed in vitro using the microsome fraction of T lymphocytes. In order to identify the phosphatases acting at these two sites, the immunoprecipitated gamma chain was used as substrate either following treatment with protein kinase C in vitro, in which case phosphorylation occurs mainly at Ser 123, or following in vivo phosphorylation of Ser 126. Purified oligomeric forms of the polycation-stimulated phosphatases were more effective in dephosphorylating both phosphorylated forms of the gamma chain compared with equivalent amounts of ATP,Mg2+-dependent phosphatases or calcineurin. By using phosphopeptide analogues of the CD3 gamma chain containing Ser 123 or Ser 126 as substrates (A3 and A6), it was shown that polycation-stimulated phosphatases selectively dephosphorylated Ser 123 compared to Ser 126. In order to determine which phosphatases dephosphorylate the gamma chain in microsomes, A3 and A6 were used as substrates for characterising phosphatases in microsomes from human T leukaemia Jurkat 6 cells. Three phosphopeptide phosphatases (250-400 kDa) co-eluted through five purification steps with three forms of polycation-stimulated phosphorylase phosphatase. The partially purified A3/A6 phosphopeptide phosphatases were insensitive to Ca2+, calmodulin and inhibitor-1, and dephosphorylated A3 preferentially compared with A6. A latent form of microsomal ATP,Mg2+-dependent phosphorylase phosphatase was stimulated 10-fold by trypsinisation, but did not dephosphorylate phosphopeptides A3 and A6. The results show that high-Mr forms of polycation-stimulated phosphatases are the only enzymes in human T leukaemia cell microsomes which dephosphorylate gamma chain phosphopeptides. The data point to an important role for polycation-stimulated phosphatases in regulating the phosphorylation state, and so function(s), of the CD3 antigen.     

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.     

Biochemical and biological characterization of a neuroendocrine-associated phosphatase

The biochemical and biological properties of a novel neuroendocrine-associated phosphatase (NEAP) were characterized. NEAP had a sequence characteristic of a dual-specificity phosphatase (DSP), and was preferentially expressed in neuroendocrine cells/tissues as well as in skeletal muscle and heart. Expression of NEAP was up-regulated in nerve growth factor (NGF)-treated, differentiated PC12 cells. NEAP was cytosolic and did not apparently have effects against extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase activated by various stimuli. Although NEAP and MAPK phosphatase (MPK)-1 showed similar phosphatase activity towards p-nitro phenylphosphate (pNPP), in contrast to MKP-1, NEAP did not dephosphorylate JNK and p38-MAPK in vitro. Overexpression of NEAP, but not the C152S mutant, in PC12 cells suppressed NGF-induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt activation. Overexpression of NEAP also suppressed neurite outgrowth induced by NGF and sensitized PC12 cells to cisplatin-induced apoptosis. Suppression of NEAP by RNA interference enhanced NGF-induced neurite outgrowth and Akt activation. Our results indicated that, unlike other DSPs, down-regulation of conventional MAPKs was not the major function of NEAP. Furthermore, NEAP might be involved in neuronal differentiation via regulation of the PI3K/Akt signaling.     

Dynamic interaction between the dual specificity phosphatase MKP7 and the JNK3 scaffold protein beta-arrestin 2

JNK scaffold proteins bind JNK and upstream kinases to activate subsets of JNK and localize activated JNK to specific subcellular sites. We previously demonstrated that the dual specificity phosphatases (DSPs) MKP7 and M3/6 bind the scaffold JNK-interacting protein-1 (JIP-1) and inactivate the bound subset of JNK (1). The G protein-coupled receptor (GPCR) adaptor beta-arrestin 2 is also a JNK3 scaffold. It binds the upstream kinases ASK1 and MKK4 and couples stimulation of the angiotensin II receptor AT1aR to activation of a cytoplasmic pool of JNK3. Here we report that MKP7 also binds beta-arrestin 2 via amino acids 394-443 of MKP7, the same region that interacts with JIP-1. This region of MKP7 interacts with beta-arrestin 2 at a central region near the JNK binding domain. MKP7 dephosphorylates JNK3 bound to beta-arrestin 2, either following activation by ASK1 overexpression or following AT1aR stimulation. Initial AT1aR stimulation causes a rapid (within 5 min) dissociation of MKP7 from beta-arrestin 2. MKP7 then reassociates with beta-arrestin 2 on endocytic vesicles 30-60 min after initial receptor stimulation. This dynamic interaction between phosphatase and scaffold permits signal transduction through a module that binds both positive and negative regulators.     

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic (32)P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.     

Protein kinase and protein phosphatase expression in the central nervous system of G93A mSOD over-expressing mice

The expressions of 78 protein kinases, 24 protein phosphatases and 31 phosphoproteins were investigated by Kinetworks trade mark analysis in brain and spinal cord tissue of transgenic mice over-expressing G93A mutant superoxide dismutase (mSOD), a murine model of amyotrophic lateral sclerosis (ALS). In the brains of affected mSOD mice, we observed increased expression of cAMP-dependent protein kinase (PKA, 111% increase compared with control), and protein phosphatase 2B Aalpha-catalytic subunit (calcineurin, 109% increase), and reductions in the levels of PAK3 (76% decrease) and protein phosphatase 2C Cbeta-subunit (32% decrease). Increased Ser259 phosphorylation of Raf1 (126% increase) in mSOD mice correlated with higher expression of p73 Raf1 (147% increase). There was also increased p73 Raf1 (69% increase) and Ser259 phosphorylation (45% increase) in the spinal cords of mSOD mice. While adducin underwent enhanced phosphorylation (alphaS724, 90% increase; gammaS662, 290% increase) in mSOD brain, its phosphorylation was lower in the mSOD spinal cord (alphaS724, 53% decrease; gammaS662, 46% decrease). In spinal cords of affected mSOD mice, we also observed elevated expression of casein kinase 1delta (CK1delta, 157% increase), JAK2 (84% increase), PKA (183% increase), protein kinase C (PKC) delta (123% increase), p124 PKC micro (142% increase), and RhoA kinase (221% increase), and enhanced phosphorylation of extracellular regulated kinases 1 (ERK1, T202/Y204, 90% increase), and 2 (ERK2, T185/Y187, 73% increase), p38 MAP kinase (T180/Y182, 1570% increase), and PKBalpha (T308, 154% increase; S473, 61% increase). There was also reduced phosphorylation of RB (S780, 45% decrease; S807/S811, 65% decrease), Src (Y418, 63% decrease) and p40 SAPK/JNKbeta (T183/Y185, 43% decrease). Variability in the expression of kinases, phosphatases and phosphorylation of their substrates was observed even in mutant animals having a similar phenotype. The expression and phosphorylation differences between mSOD and control mice were dissimilar to those between ALS patients and controls. This finding indicates that the activation of protein kinases and phosphoproteins is different with neuron loss in the mSOD mouse compared with that seen in patients with the sporadic form of ALS.     

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G₂/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G₂/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G₂/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.     

JNK1 Phosphorylates SIRT1 and Promotes Its Enzymatic Activity

SIRT1 is a NAD-dependent deacetylase that regulates a variety of pathways including the stress protection pathway. SIRT1 deacetylates a number of protein substrates, including histones, FOXOs, PGC-1α, and p53, leading to cellular protection. We identified a functional interaction between cJUN N-terminal kinase (JNK1) and SIRT1 by coimmunoprecipitation of endogenous proteins. The interaction between JNK1 and SIRT1 was identified under conditions of oxidative stress and required activation of JNK1 via phosphorylation. Modulation of SIRT1 activity or protein levels using nicotinamide or RNAi did not modify JNK1 activity as measured by its ability to phosphorylate cJUN. In contrast, human SIRT1 was phosphorylated by JNK1 on three sites: Ser27, Ser47, and Thr530 and this phosphorylation of SIRT1 increased its nuclear localization and enzymatic activity. Surprisingly, JNK1 phosphorylation of SIRT1 showed substrate specificity resulting in deacetylation of histone H3, but not p53. These findings identify a mechanism for regulation of SIRT1 enzymatic activity in response to oxidative stress and shed new light on its role in the stress protection pathway.     

Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation

It has been demonstrated previously that during mitosis the sites of myosin phosphorylation are switched between the inhibitory sites, Ser 1/2, and the activation sites, Ser 19/Thr 18 (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129- 137; Satterwhite, L.L., M.J. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), suggesting a regulatory role of myosin phosphorylation in cell division. To explore the function of myosin phosphatase in cell division, the possibility that myosin phosphatase activity may be altered during cell division was examined. We have found that the myosin phosphatase targeting subunit (MYPT) undergoes mitosis-specific phosphorylation and that the phosphorylation is reversed during cytokinesis. MYPT phosphorylated either in vivo or in vitro in the mitosis-specific way showed higher binding to myosin II (two- to threefold) compared to MYPT from cells in interphase. Furthermore, the activity of myosin phosphatase was increased more than twice and it is suggested this reflected the increased affinity of myosin binding. These results indicate the presence of a unique positive regulatory mechanism for myosin phosphatase in cell division. The activation of myosin phosphatase during mitosis would enhance dephosphorylation of the myosin regulatory light chain, thereby leading to the disassembly of stress fibers during prophase. The mitosis-specific effect of phosphorylation is lost on exit from mitosis, and the resultant increase in myosin phosphorylation may act as a signal to activate cytokinesis.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.     

p21-activated kinase 2 in neutrophils can be regulated by phosphorylation at multiple sites and by a variety of protein phosphatases

The p21-activated kinase(Pak) 2 undergoes rapid autophosphorylation/activation in neutrophils stimulated with a variety of chemoattractants (e.g., fMLP). Phosphorylation within the activation loop (Thr(402)) and inhibitory domain (Ser(141)) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser(20)) and Pak-interacting guanine nucleotide exchange factor (Ser(192) and Ser(197)) binding sites blocks the interactions of Pak 2 with these proteins. A panel of phosphospecific Abs was used to investigate the phosphorylation of Pak 2 in neutrophils at these sites. Pak 2 underwent rapid (< or =15 s) phosphorylation at Ser(20), Ser(192/197), and Thr(402) in neutrophils stimulated with fMLP. Phosphorylation at Ser(192/197) and Thr(402) were highly transient events, whereas that at Ser(20) was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser(141) in unstimulated neutrophils and phosphorylation at this site was less sensitive to cell stimulation than at other residues. Studies with selective inhibitors suggested that a variety of phosphatases might be involved in the rapid dephosphorylation of Pak 2 at Thr(402) in stimulated neutrophils. This was consistent with biochemical studies which showed that the activation loop of GST-Pak 3, which is homologous to that in Pak 2, was a substrate for protein phosphatase 1, 2A, and a Mg(2+)/Mn(2+)-dependent phosphatase(s) which exhibited properties different from those of the conventional isoforms of protein phosphatase 2C. The data indicate that Pak 2 undergoes a complex pattern of phosphorylation in neutrophils and that dephosphorylation at certain sites may involve multiple protein phosphatases that exhibit distinct modes of regulation.     

A phosphatase‐centric mechanism drives stress signaling response

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase‐driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho‐proteomic response is the Endosulfine‐mediated inhibition of protein phosphatase 2A‐Cdc55 (PP2A^Cdc55), while we do not observe concurrent kinase activation. In fact, many of the stress‐induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2A^Cdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well‐established kinase‐centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re‐evaluating the impact of phosphatases on shaping the phosphorylome.