A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase

Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser(231) residue, located within the KIM. Upon phosphorylation of Ser(231), PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal-regulated kinase (ERK)1/2 and p38alpha were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Calpha catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38alpha by wild-type PTP-SL, but not by a PTP-SL S231A mutant. These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases.     

Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2

Activating mutations within the K-ras gene occur in a high percentage of human pancreatic carcinomas. We reported previously that the presence of oncogenic, activated K-ras in human pancreatic carcinoma cell lines did not result in constitutive activation of the extracellular signal-regulated kinases (ERK1 and ERK2). In the present study, we further characterized the ERK signaling pathway in pancreatic tumor cell lines in order to determine whether the ERK pathway is subject to a compensatory downregulation. We found that the attenuation of serum-induced ERK activation was not due to a delay in the kinetics of ERK phosphorylation. Treatment with the tyrosine phosphatase inhibitor orthovanadate increased the level of ERK phosphorylation, implicating a vanadate-sensitive tyrosine phosphatase in the negative regulation of ERK. Furthermore, expression of a dual specificity phosphatase capable of inactivating ERK known as mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2) was elevated in most of the pancreatic tumor cell lines and correlated with the presence of active MAP kinase kinase (MEK). Taken together, these results suggest that pancreatic tumor cells expressing oncogenic K-ras compensate, in part, by upregulating the expression of MKP-2 to repress the ERK signaling pathway.     

Cellular adhesion of primary Sertoli cells affects responsiveness of the extracellular signal-regulated kinases 1 and 2 to follicle-stimulating hormone but not to epidermal growth factor

The cellular adhesion status and the exposure to soluble growth factors both contribute to mitogen-activated protein (MAP) kinase activation. To date, however, whether mitogens acting through G-protein-coupled receptors (GPCRs) need cell adhesion to activate MAP kinases/extracellular signal-regulated kinases (ERK) 1, 2 has been poorly investigated. We addressed this point in primary cultures of Sertoli cells experimentally maintained in suspension, considering that follicle-stimulating hormone (FSH) activates ERK1, 2 in attached Sertoli cells by acting through a GPCR. We found that FSH actively repressed ERK1, 2, in a cAMP-dependent but cAMP-dependent protein kinase (PKA)-independent manner, and this inhibition required the activity of a tyrosine phosphatase. In comparison, in the absence of anchorage, ERK1, 2 were still activated by epidermal growth factor, in a PKA-dependent manner. Altogether, these data suggest that sensitivity of the MAP kinase response toward cell adhesion may depend, at least in part, on the class of receptor, GPCR or receptor with tyrosine kinase activity, by which it is triggered.     

Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies.

A protein kinase characterized by its ability to phosphorylate microtubule-associated protein-2 (MAP2) and myelin basic protein (MBP) is thought to play a pivotal role in the transduction of signals from many receptors in response to their ligands. A kinase with such activity, named extracellular signal-regulated kinase 1 (ERK1), is activated rapidly by numerous extracellular signals, requires phosphorylation on tyrosine to be fully active, and in vitro can activate a kinase (a ribosomal S6 protein kinase) that is downstream in phosphorylation cascades. From the protein sequence predicted by the rat ERK1 cDNA, peptides were synthesized and used to elicit antibodies. The antibodies recognize both ERK1; a closely related kinase, ERK2; and a third novel ERK-related protein. Using these antibodies we have determined that ERK1 and ERK2 are ubiquitously distributed in rat tissues. Both enzymes are expressed most highly in brain and spinal cord as are their mRNAs. The third ERK protein was found in spinal cord and in testes. The antibodies detect ERKs in cell lines from multiple species, including human, mouse, dog, chicken, and frog, in addition to rat, indicating that the kinases are conserved across species. ERK1 and ERK2 have been separated by chromatography on Mono Q. Stimulation by insulin increases the phosphorylation of both kinases on tyrosine residues, as assessed by immunoblotting with phosphotyrosine antibodies, and retards their elution from Mono Q. Each of these ERKs appears to account for a distinct peak of MBP kinase activity. The activity in each peak is diminished by incubation with either phosphatase 2a or CD45. Therefore, both enzymes have similar modes of regulation and appear to contribute to the growth factor-stimulated MAP2/MBP kinase activity measured in cell extracts.     

Distinctive features of Trk neurotrophin receptor transactivation by G protein-coupled receptors.

Ligands for G protein-coupled receptors (GPCR) are capable of activating mitogenic receptor tyrosine kinases, in addition to the mitogen-activated protein (MAP) kinase signaling pathway and classic G protein-dependent signaling pathways involving adenylyl cyclase and phospholipase. For example, receptors for epidermal growth factor (EGF), insulin-like growth-1 and platelet-derived growth factor and can be transactivated through G protein-coupled receptors. Neurotrophins, such as NGF, BDNF and NT-3 also utilize receptor tyrosine kinases, namely TrkA, TrkB and TrkC. Recently, it has been shown that activation of Trk receptor tyrosine kinases can also occur via a G protein-coupled receptor mechanism, without involvement of neurotrophins. Adenosine and adenosine agonists can activate Trk receptor phosphorylation specifically through the seven transmembrane spanning adenosine 2A (A2A) receptor. Several features of Trk receptor transactivation are noteworthy and differ significantly from other transactivation events. Trk receptor transactivation is slower and results in a selective increase in activated Akt. Unlike the biological actions of other tyrosine kinase receptors, increased Trk receptor activity by adenosine resulted in increased cell survival. This article will discuss potential mechanisms by which adenosine can activate trophic responses through Trk tyrosine kinase receptors.     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation.

Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.     

Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages.

Bacterial lipopolysaccharide (LPS) is a potent activator of antibacterial responses by macrophages. Following LPS stimulation, the tyrosine phosphorylation of several proteins is rapidly increased in macrophages, and this event appears to mediate some responses to LPS. We now report that two of these tyrosine phosphoproteins of 41 and 44 kDa are isoforms of mitogen-activated protein (MAP) kinase. Each of these proteins was reactive with anti-MAP kinase antibodies and comigrated with MAP kinase activity in fractions eluted from a MonoQ anion-exchange column. Following LPS stimulation, column fractions containing the tyrosine phosphorylated forms of p41 and p44 exhibited increased MAP kinase activity. Inhibition of LPS-induced tyrosine phosphorylation of these proteins was accompanied by inhibition of MAP kinase activity. Additionally, induction of p41/p44 tyrosine phosphorylation and MAP kinase activity by LPS appeared to be independent of activation of protein kinase C, even though phorbol esters also induced these responses. These results demonstrate that LPS induces the tyrosine phosphorylation and activation of at least two MAP kinase isozymes. Since MAP kinases appear to modulate cellular processes in response to extracellular signals, these kinases may be important targets for LPS action in macrophages.     

Activation of the MAP kinase pathway by the protein kinase raf.

Both MAP kinases and the protein kinase p74raf-1 are activated by many growth factors in a c-ras-dependent manner and by oncogenic p21ras. We were therefore interested in determining the relationship between MAP kinases and raf. The MAP kinase ERK2 is activated by expression of oncogenically activated raf, independently of cellular ras. Overexpressed p74raf-1 potentiates activation of ERK2 by EGF and TPA. MAP kinase kinase inactivated by phosphatase 2A treatment is phosphorylated and reactivated by incubation with p74raf-1 immunoprecipitated from phorbol ester-treated cells. We conclude that raf protein kinase is upstream of MAP kinases and is either a MAP kinase kinase kinase or a MAP kinase kinase kinase kinase.     

Different protein kinase families are activated by osmotic stresses in Arabidopsis thaliana cell suspensions. Involvement of the MAP kinases AtMPK3 and AtMPK6

Five Ca(2+)-independent protein kinases were rapidly activated by hypoosmotic stress, moderate or high hyperosmolarity induced by several osmolytes, sucrose, mannitol or NaCl. Three of these kinases, transiently activated by hypoosmolarity, recognised by anti-phosphorylated mitogen-activated protein (MAP) kinase antibodies, sensitive to a MAP kinase inhibitor and inactivated by the action of a tyrosine phosphatase, corresponded to MAP kinases. Using specific antibodies, two of the MAP kinases were identified as AtMPK6 and AtMPK3. The two other protein kinases, durably activated by high hyperosmolarity, did not belong to the MAP kinase family. Activation of AtMPK6 and AtMPK3 by hypoosmolarity depended on upstream protein kinases sensitive to staurosporine and on calcium influx. In contrast, these two transduction steps were not involved in the activation of the two protein kinases activated by high hyperosmolarity.     

Cbl: many adaptations to regulate protein tyrosine kinases

Responses to extracellular stimuli are often transduced from cell-surface receptors to protein tyrosine kinases which, when activated, initiate the formation of protein complexes that transmit signals throughout the cell. A prominent component of these complexes is the product of the proto-oncogene c-Cbl, which specifically targets activated protein tyrosine kinases and regulates their signalling. How, then, does this multidomain protein shape the responses generated by these signalling complexes?     

Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts.

BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect of inactivation of the c-src gene. In response to adhesion on a fibronectin substrate, RPTPalpha-/- fibroblasts also exhibited characteristic deficiencies in integrin-mediated signalling responses, such as decreased tyrosine phosphorylation of the c-Src substrates Fak and p 130(cas), and reduced activation of extracellular signal regulated (Erk) MAP kinases. CONCLUSIONS: These observations demonstrate that RPTPalpha functions as a physiological upstream activator of Src-family kinases in fibroblasts and establish this tyrosine phosphatase as a newly identified regulator of integrin signalling.     

Reduced activation and expression of ERK1/2 MAP kinase in the post-mortem brain of depressed suicide subjects

The extracellular regulated kinases (ERK) 1 and ERK2 are members of mitogen-activated protein (MAP) kinase family that play an important role in transducing extracellular signals to the nucleus and have been implicated in a broad spectrum of biological responses. To test the hypothesis that MAP kinases may be involved in depression, we examined the activation of p44/42 MAP kinase and expression of ERK1 and ERK2 in the post-mortem brain tissue obtained from non-psychiatric control subjects (n = 11) and age- and the post-mortem interval-matched depressed suicide subjects (n = 11). We observed that p44/42 MAP kinase activity was significantly decreased in the prefrontal cortical areas (Brodmann's areas 8, 9 and 10) and the hippocampus of depressed suicide subjects without any change in the cerebellum. This decrease was associated with a decrease in mRNA and protein levels of ERK1 and ERK2. In addition, the expression of MAP kinase phosphatase (MKP)2, a 'dual function' ERK1/2 phosphatase, was increased in the prefrontal cortex and hippocampus. These studies suggest that p44/42 MAP kinases are less activated in the post-mortem brain of depressed suicide subjects and this may be because of reduced expression of ERK1/2 and increased expression of MKP2. Given the role of MAP kinases in various physiological functions and gene expression, alterations in p44/42 MAP kinase activation and expression of ERK1/2 may contribute significantly to the pathophysiology of depressive disorders.     

Identification of extracellularly phosphorylated membrane proteins

Ecto‐protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC‐MS/MS. A “false‐positive” strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G‐protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface‐exposed peptides when extracellular adenosine‐5'‐triphosphate is elevated during purinergic signalling.     

Protein phosphatase 2A forms a molecular complex with Shc and regulates Shc tyrosine phosphorylation and downstream mitogenic signaling

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.     

Selective down-regulation of angiotensin II receptor type 1A signaling by protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells

The heptahelical AT(1) G-protein-coupled receptor lacks inherent tyrosine kinase activity. Angiotensin II binding to AT(1) nevertheless activates several tyrosine kinases and stimulates both tyrosine phosphorylation and phosphatase activity of the SHP-2 tyrosine phosphatase in vascular smooth muscle cells. Since a balance between tyrosine kinase and tyrosine phosphatase activities is essential in angiotensin II signaling, we investigated the role of SHP-2 in modulating tyrosine kinase signaling pathways by stably transfecting vascular smooth muscle cells with expression vectors encoding wild-type SHP-2 protein or a catalytically inactive SHP-2 mutant. Our data indicate that SHP-2 is an efficient negative regulator of angiotensin II signaling. SHP-2 inhibited c-Src catalytic activity by dephosphorylating a positive regulatory tyrosine 418 within the Src kinase domain. Importantly, SHP-2 expression also abrogated angiotensin II-induced activation of ERK, whereas expression of catalytically inactive SHP-2 caused sustained ERK activation. Thus, SHP-2 likely regulates angiotensin II-induced MAP kinase signaling by inactivating c-Src. These SHP-2 effects were specific for a subset of angiotensin II signaling pathways, since SHP-2 overexpression failed to influence Jak2 tyrosine phosphorylation or Fyn catalytic activity. These data show SHP-2 represents a critical negative regulator of angiotensin II signaling, and further demonstrate a new function for this phosphatase in vascular smooth muscle cells.     

Roles of cysteines in rat dipeptidyl peptidase IV/CD26 in processing and proteolytic activity.

The multifunctional type II transmembrane glycoprotein, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), is expressed by almost all mammalian cells and is identical to the adenosine deaminase binding protein CD26 on lymphocytes. The extracellular part of rat DPPIV can be divided into three domains the middle part of which harbors 10 of the 12 highly conserved cysteine residues. The cysteine-rich domain is responsible for DPPIV-binding to collagen I and to extracellular ADA. The participation of distinct cysteines in disulfide bridges is not yet known. Titration experiments have shown the presence of six free cysteines and three disulfide bridges in native rat DPPIV. To investigate the role of distinct cysteines in the structure-function relationships of rat DPPIV we constructed 12 different cysteine point mutations (C299, C326, C383, C455, C650 mutated to G; C337, C395, C445, C448, C473, C552, C763 mutated to S). Intracellular translocation to the cell surface of stable transfected Chinese hamster ovary cells was examined with antibodies against different epitopes of DPPIV. Surface expression of mutants C326G, C445S and C448S is inhibited totally; mutants C337S, C455G, C473S and C552S show weak expression only. In parallel, the half-life of these mutants is reduced to < 10% compared with wild-type enzyme. We were able to show that the specific peptidase activity of the mutant protein depends on cell-surface expression, dimerization and the existence of a 150-kDa form demonstrable by nondenaturing SDS/PAGE. We conclude that cysteine residues 326, 337, 445, 448, 455, 473 and 552 in rat DPPIV are essential for the correct folding and intracellular trafficking of this glycoprotein, and therefore for its normal biological properties.     

Enhancement of TNF-alpha-induced apoptosis by immobilized arginine-glycine-aspartate: involvement of a tyrosine kinase-dependent, MAP kinase-independent mechanism

Extracellular matrix facilitates anchorage-dependent cell survival via interaction of its arginine-glycine-aspartate (RGD) motif with integrins. In this report, we describe an unexpected, apoptosis-promoting the effect of immobilized RGD (iRGD) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Mesangial cells cultured on RGD-coated plates showed enhanced susceptibility to TNF-alpha-induced apoptosis. iRGD alone did not affect cell survival. In contrast, iRGD did not facilitate but inhibited apoptosis induced by H(2)O(2). Mitogen-activated protein (MAP) kinases and tyrosine kinases are important mediators for the RGD-integrin signaling. Pretreatment with MAP kinase kinase inhibitor PD098059, c-Jun N-terminal kinase (JNK)-c-Jun/AP-1 inhibitor curcumin or p38 MAP kinase inhibitor SB203580 did not attenuate the apoptosis-promoting effect of iRGD. Consistently, transfection with dominant-negative mutants of extracellular signal-regulated kinases, JNK or p38 MAP kinase did not inhibit the effect of iRGD. In contrast, protein tyrosine kinase inhibitors, genistein, and herbimycin A, abrogated the apoptosis-promoting effect of iRGD. Of note, TNF-alpha-induced apoptosis on uncoated plates was not attenuated by tyrosine kinase inhibitors. These data provide the first evidence that iRGD accelerates certain apoptosis. We identified that the effect was mediated by the tyrosine kinase-dependent, MAP kinase-independent mechanism.