Negative regulation of PYK2/related adhesion focal tyrosine kinase signal transduction by hematopoietic tyrosine phosphatase SHPTP1.

Related adhesion focal tyrosine kinase (RAFTK) (also known as PYK2) is a cytoplasmic tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). RAFTK is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha, changes in osmolarity, elevation in intracellular calcium concentration, lysophosphatidic acid, and bradykinin. Overexpression of RAFTK induces activation of c-Jun amino-terminal kinase (also known as stress-activated protein kinase), mitogen-activated protein kinase (MAPK), and p38 MAPK. The present studies demonstrate that RAFTK binds constitutively to the protein tyrosine phosphatase SHPTP1. In contrast to PTP1B, overexpression of wild-type SHPTP1 blocks tyrosine phosphorylation of RAFTK. The results further demonstrate that RAFTK is a direct substrate of SHPTP1 in vitro. Moreover, treatment of PC12 cells with bradykinin is associated with inhibition in tyrosine phosphorylation of RAFTK in the presence of SHPTP1. Furthermore, in contrast to the phosphatase-dead SHPTP1 C453S mutant, overexpression of wild-type SHPTP1 blocks interaction of RAFTK with the SH2-domain of c-Src and inhibits RAFTK-mediated MAPK activation. Significantly, cotransfection of RAFTK with SHPTP1 did not inhibit RAFTK-mediated c-Jun amino-terminal kinase activation. Taken together, these findings suggest that SHPTP1 plays a negative role in PYK2/RAFTK signaling by dephosphorylating RAFTK.     

Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.     

The SH2 domain containing tyrosine phosphatase-1 down-regulates activation of Lyn and Lyn-induced tyrosine phosphorylation of the CD19 receptor in B cells

SHP-1 is a cytosolic tyrosine phosphatase implicated in down-regulation of B cell antigen receptor signaling. SHP-1 effects on the antigen receptor reflect its capacity to dephosphorylate this receptor as well as several inhibitory comodulators. In view of our observation that antigen receptor-induced CD19 tyrosine phosphorylation is constitutively increased in B cells from SHP-l-deficient motheaten mice, we investigated the possibility that CD19, a positive modulator of antigen receptor signaling, represents another substrate for SHP-1. However, analysis of CD19 coimmunoprecipitable tyrosine phosphatase activity in CD19 immunoprecipitates from SHP-1-deficient and wild-type B cells revealed that SHP-1 accounts for only a minor portion of CD19-associated tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is modulated by the Lyn protein-tyrosine kinase, Lyn activity was evaluated in wild-type and motheaten B cells. The results revealed both Lyn as well as CD19-associated Lyn kinase activity to be constitutively and inducibly increased in SHP-1-deficient compared with wild-type B cells. The data also demonstrated SHP-1 to be associated with Lyn in stimulated but not in resting B cells and indicated this interaction to be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These findings, together with cyanogen bromide cleavage data revealing that SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling.     

SHP-1 requires inhibitory co-receptors to down-modulate B cell antigen receptor-mediated phosphorylation of cellular substrates

Signaling through the B cell antigen receptor (BCR) is negatively regulated by the SH2 domain-containing protein-tyrosine phosphatase SHP-1, which requires association with tyrosine-phosphorylated proteins for activation. Upon BCR ligation, SHP-1 has been shown to associate with the BCR, the cytoplasmic protein-tyrosine kinases Lyn and Syk, and the inhibitory co-receptors CD22 and CD72. How SHP-1 is activated by BCR ligation and regulates BCR signaling is, however, not fully understood. Here we demonstrate that, in the BCR-expressing myeloma line J558L mu 3, CD72 expression reduces the BCR ligation-induced phosphorylation of the BCR component Ig alpha/Ig beta and its cytoplasmic effectors Syk and SLP-65. Substrate phosphorylation was restored by expression of dominant negative mutants of SHP-1, whereas the SHP-1 mutants failed to enhance phosphorylation of the cellular substrates in the absence of CD72. This indicates that SHP-1 is efficiently activated by CD72 but not by other pathways in J558L mu m3 cells and that inhibition of SHP-1 specifically activated by CD72 reverses CD72-induced dephosphorylation of cellular substrates in these cells. Taken together, BCR-induced SHP-1 activation is likely to require inhibitory co-receptors such as CD72, and SHP-1 appears to mediate the negative regulatory effect of CD72 on BCR signaling by dephosphorylating Ig alpha/Ig beta and its downstream signaling molecules Syk and SLP-65.     

Fyn kinase-directed activation of SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi protein-coupled receptors in Madin-Darby canine kidney cells

SHP-2, an SH2 domain-containing protein-tyrosine phosphatase, plays an important role in receptor tyrosine kinase-regulated cell proliferation and differentiation. Little is known about the activation mechanisms and the participation of SHP-2 in the activity of G protein-coupled receptors lacking intrinsic tyrosine kinase activity. We show that the activity of SHP-2 (but not SHP-1) is specifically stimulated by the selective alpha2A-adrenergic receptor agonist UK14304 and by lysophosphatidic acid (LPA) in Madin-Darby canine kidney (MDCK) cells. UK14304 and LPA promote the tyrosine phosphorylation of SHP-2 and its association with Grb2. The agonist-induced direct interaction of Grb2 with SHP-2 is mediated by the SH2 domain of Grb2 and the tyrosine phosphorylation of SHP-2. Rapid activation of Src family kinase by UK14304 preceded the SHP-2 activation. Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in MDCK cells, Fyn was the only one specifically associated with SHP-2, and the physical interaction between them, which requires the Src family kinase activity, was increased in response to the agonists. Pertussis toxin, PP1 (a selective Src family kinase inhibitor), or overexpression of a catalytically inactive mutant of Fyn blocked the UK14304- or LPA-stimulated activity of SHP-2, SHP-2 tyrosine phosphorylation, and SHP-2 association with Grb2. Therefore, we have demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors requires Fyn kinase and that there is a specific physical interaction of Fyn kinase with SHP-2 in MDCK cells.     

Activation of SAPK/JNK signaling by protein kinase Cdelta in response to DNA damage

The cellular response to genotoxic stress includes activation of protein kinase Cdelta (PKCdelta). The functional role of PKCdelta in the DNA damage response is unknown. The present studies demonstrate that PKCdelta is required in part for induction of the stress-activated protein kinase (SAPK/JNK) in cells treated with 1-beta-d-arabinofuranosylcytosine (araC) and other genotoxic agents. DNA damage-induced SAPK activation was attenuated by (i) treatment with rottlerin, (ii) expression of a kinase-inactive PKCdelta(K-R) mutant, and (iii) down-regulation of PKCdelta by small interfering RNA (siRNA). Coexpression studies demonstrate that PKCdelta activates SAPK by an MKK7-dependent, SEK1-independent mechanism. Previous work has shown that the nuclear Lyn tyrosine kinase activates the MEKK1 --> MKK7 --> SAPK pathway but not through a direct interaction with MEKK1. The present results extend those observations by demonstrating that Lyn activates PKCdelta, and in turn, MEKK1 is activated by a PKCdelta-dependent mechanism. These findings indicate that PKCdelta functions in the activation of SAPK through a Lyn --> PKCdelta --> MEKK1 --> MKK7 --> SAPK signaling cascade in response to DNA damage.     

Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.     

Role for Lyn tyrosine kinase as a regulator of stress-activated protein kinase activity in response to DNA damage

The cellular response to DNA damage includes activation of the nuclear Lyn protein tyrosine kinase. Using cells deficient in Lyn expression, the present studies demonstrate that Lyn is required in part for induction of the stress-activated protein kinase (SAPK) in the response to 1-beta-D-arabinofuranosylcytosine (ara-C) and other genotoxic agents. By contrast, exposure of Lyn-deficient cells to ara-C, ionizing radiation, or cisplatin had no effect on activation of extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase. Similar findings were obtained in cells stably expressing a kinase-inactive, dominant-negative Lyn(K-R) mutant. Coexpression studies demonstrate that Lyn, but not Lyn(K-R), induces SAPK activity. In addition, the results demonstrate that Lyn activates SAPK by an MKK7-dependent, SEK1-independent mechanism. As MEKK1 functions upstream to MKK7 and SAPK, the finding that a dominant-negative MEKK1(K-M) mutant blocks Lyn-induced SAPK activity supports involvement of the MEKK1-->MKK7 pathway. The results also demonstrate that inhibition of Lyn-induced SAPK activity abrogates the apoptotic response of cells to genotoxic stress. These findings indicate that activation of SAPK by DNA damage is mediated in part by Lyn and that the Lyn-->MEKK1-->MKK7-->SAPK pathway is functional in the induction of apoptosis by genotoxic agents.     

Consequences of direct versus indirect activation of epidermal growth factor receptor in intestinal epithelial cells are dictated by protein-tyrosine phosphatase 1B

The epidermal growth factor receptor (EGFR) is an integral regulator of many cellular functions. EGFR also acts as a central conduit for extracellular signals involving direct activation of the receptor by EGFR ligands or indirect activation by G protein-coupled receptor (GPCR)-stimulated transactivation of the EGFR. We have previously shown that EGFR negatively regulates epithelial chloride secretion as a result of transforming growth factor-alpha-mediated EGFR transactivation in response to muscarinic GPCR activation. Here we show that direct activation of the EGFR by EGFR ligands produces a different pattern of EGFR tyrosine phosphorylation and downstream phosphatidylinositol 3-kinase recruitment than GPCR-stimulated transactivation of the EGFR occurring via paracrine EGFR ligand release. Moreover, we demonstrate that this differential signaling and its consequences depend on protein-tyrosine phosphatase 1B activity. Thus protein-tyrosine phosphatase 1B governs differential recruitment of signaling pathways involved in EGFR regulation of epithelial ion transport. Our findings furthermore establish how divergent signaling outcomes can arise from the activation of a single receptor.     

The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B.

The protein-tyrosine phosphatase PTP-1B is an important regulator of intracellular protein tyrosine phosphorylation, and is itself regulated by phosphorylation. We report that PTP-1B and its yeast analog, YPTP, are phosphorylated and activated by members of the CLK family of dual specificity kinases. CLK1 and CLK2 phosphorylation of PTP-1B in vitro activated the phosphatase activity approximately 3-5-fold using either p-nitrophenol phosphate, or tyrosine-phosphorylated myelin basic protein as substrates. Co-expression of CLK1 or CLK2 with PTP-1B in HEK 293 cells led to a 2-fold stimulation of phosphatase activity in vivo. Phosphorylation of PTP-1B at Ser(50) by CLK1 or CLK2 is responsible for its enzymatic activation. These findings suggest that phosphorylation at Ser(50) by serine threonine kinases may regulate the activation of PTP-1B in vivo. We also show that CLK1 and CLK2 phosphorylate and activate the S. cerevisiae PTP-1B family member, YPTP1. CLK1 phosphorylation of YPTP1 led to a 3-fold stimulation of phosphatase activity in vitro. We demonstrate that CLK phosphorylation of Ser(83) on YPTP1 is responsible for the activation of this enzyme. These findings demonstrate that the CLK kinases activate PTP-1B family members, and this phosphatase may be an important cellular target for CLK action.     

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.     

Bcl-xL blocks activation of related adhesion focal tyrosine kinase/proline-rich tyrosine kinase 2 and stress-activated protein kinase/c-Jun N-terminal protein kinase in the cellular response to methylmethane sulfonate

The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade.     

Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases.

We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo.     

Negative regulation of HER2 signaling by the PEST-type protein-tyrosine phosphatase BDP1

Signaling by receptor tyrosine kinases (RTK) mediates a variety of complex cellular functions and in case of deregulation can contribute to pathophysiological processes. A tight and finely tuned control of RTK activity is therefore critical for the cell. We investigated the role of the PEST-type protein-tyrosine phosphatase BDP1 in the regulation of HER2, a member of the epidermal growth factor receptor (EGFR) family of RTKs. Here we demonstrate that HER2 signaling is highly sensitive to BDP1 activity. Overexpression of BDP1 inhibited ligand-induced activation of HER2 but not that of the closely related EGFR. On the other hand, suppression of endogenous BDP1 expression increased the phosphorylation state of HER2. In addition, BDP1 was able to interfere with downstream signaling events by inhibiting the phosphorylation of the adaptor protein Gab1 and reducing mitogen-activated protein kinase activation. Supported by the finding that BDP1 is coexpressed with HER2 in breast cancer cells, we suggest that BDP1 is an important regulator of HER2 activity and thus the first protein-tyrosine phosphatase shown to be involved in HER2 signal attenuation.     

Identification of new substrates of the protein-tyrosine phosphatase PTP1B by Bayesian integration of proteome evidence

There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzyme-substrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and "closeness" in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-γ1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation.     

Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling

The glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex, a key activatory receptor for collagen on platelet surface membranes, is constitutively associated with the Src family kinases Fyn and Lyn. Molecular cloning of GPVI has revealed the presence of a proline-rich domain in the sequence of GPVI cytoplasmic tail which has the consensus for interaction with the Src homology 3 (SH3) domains of Fyn and Lyn. A series of in vitro experiments demonstrated the ability of the SH3 domains of both Src kinases to bind the proline-rich domain of GPVI. Furthermore, depletion of the proline-rich domain in GPVI (Pro(-)-GPVI) prevented binding of Fyn and Lyn and markedly reduced phosphorylation of FcR gamma-chain in transiently transfected COS-7 cells, but did not affect the association of the gamma-chain with GPVI. Jurkat cells stably transfected with wild type GPVI show robust increases in tyrosine phosphorylation and intracellular Ca2+ in response to the snake venom convulxin that targets GPVI. Importantly, convulxin is not able to activate cells transfected with Pro(-)-GPVI, even though the association with the immunoreceptor tyrosine-based activation motif-containing chains is maintained. These findings demonstrate that the proline-rich domain of GPVI mediates the association with Fyn/Lyn via their SH3 domain and that this interaction initiates activation signals through GPVI.     

Involvement of the membrane distal catalytic domain in pervanadate-induced tyrosine phosphorylation of receptor protein-tyrosine phosphatase alpha

Receptor protein-tyrosine phosphatase alpha, RPTPalpha, is a typical transmembrane protein-tyrosine phosphatase (PTP) with two cytoplasmic catalytic domains. RPTPalpha became strongly phosphorylated on tyrosine upon treatment of cells with the PTP inhibitor pervanadate. Surprisingly, mutation of the catalytic site Cys in the membrane distal PTP domain (D2), but not of the membrane proximal PTP domain (D1) that harbors the majority of the PTP activity, almost completely abolished pervanadate-induced tyrosine phosphorylation. Pervanadate-induced RPTPalpha tyrosine phosphorylation was not restricted to Tyr789, a known phosphorylation site. Cotransfection of wild-type RPTPalpha did not potentiate tyrosine phosphorylation of inactive RPTPalpha-C433SC723S, suggesting that RPTPalpha-mediated activation of kinase(s) does not underlie the observed effects. Mapping experiments indicated that pervanadate-induced tyrosine phosphorylation sites localized predominantly, but not exclusively, to the C-terminus. Our results demonstrate that RPTPalpha-D2 played a role in pervanadate-induced tyrosine phosphorylation of RPTPalpha, which may suggest that RPTPalpha-D2 is involved in protein-protein interactions.     

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

Requirement for CD45 in fine-tuning mast cell responses mediated by different ligand–receptor systems

The receptor-like protein tyrosine phosphatase CD45, the most abundant cell surface phosphatase on all nucleated hemopoietic cells, is a critical regulator of the activation status of Src family kinases (SFKs). To study the impact of CD45 on mast cell function, we compare bone marrow-derived mast cells (BMMCs) from CD45-deficient mice and from mice expressing an activating point mutation (E613R) in the juxtamembrane wedge of CD45. In response to Ag-triggered FcεR1-mediated activation, CD45-deficient BMMCs exhibit increased inhibitory Lyn phosphorylation and drastically reduced effector functions (degranulation and cytokine secretion). In contrast, CD45 E613R BMMCs show stronger effector functions after Ag-triggering than wild-type (WT) BMMCs. Despite these dichotomous phenotypes, phosphorylation of the inhibitory tyrosine in the SFK Lyn of CD45 E613R BMMCs is comparable to CD45-deficient BMMCs. This unexpected phenotype most likely is due to attenuated interaction between CD45 E613R and Lyn and a hyper-activation of the Fyn-regulated phosphatidylinositol-3-kinase pathway. Interestingly, depending on the receptor system addressed, CD45-deficient and CD45 E613R BMMCs show uniform phenotypes as well. Proliferation of both cell types in response to IL-3 and/or SF is enhanced compared to WT BMMCs. Together, the data indicate that CD45 plays a complex and essential role in fine-tuning mast cell responses mediated by different ligand–receptor systems.     

Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1.

SHPTP1 (PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. A frameshift mutation in the SHPTP1 gene causes the motheaten (me/me) mouse. These mice are essentially SHPTP1 null and display multiple hematopoietic abnormalities, most prominently hyperproliferation and inappropriate activation of granulocytes and macrophages. The me/me phenotype suggests that SHPTP1 negatively regulates macrophage proliferative pathways. Using primary bone marrow-derived macrophages from me/me mice and normal littermates, we examined the role of SHPTP1 in regulating signaling by the major macrophage mitogen colony-stimulating factor 1 (CSF-1) (also known as macrophage colony-stimulating factor). Macrophages from me/me mice hyperproliferate in response to CSF-1. In the absence of SHPTP1, the CSF-1 receptor (CSF-1R) is hyperphosphorylated upon CSF-1 stimulation, suggesting that SHPTP1 dephosphorylates the CSF-1R. At least some CSF-1R-associated proteins also are hyperactivated. SHPTP1 is associated constitutively, via its SH2 domains, with an unidentified 130-kDa phosphotyrosyl protein (P130). P130 and SHPTP1 are further tyrosyl phosphorylated upon CSF-1 stimulation. Tyrosyl-phosphorylated SHPTP1 binds to Grb2 via the Grb2 SH2 domain. Moreover, in me/me macrophages, Grb2 is associated, via its SH3 domains, with several tyrosyl phosphoproteins. These proteins are hyperphosphorylated on tyrosyl residues in me/me macrophages, suggesting that Grb2 may recruit substrates for SHPTP1. Our results indicate that SHPTP1 is a critical negative regulator of CSF-1 signaling in vivo and suggest a potential new function for Grb2.