Phosphorylation of a Src kinase at the autophosphorylation site in the absence of Src kinase activity

Exposure of cells to oxidants increases the phosphorylation of the Src family tyrosine protein kinase Lck at Tyr-394, a conserved residue in the activation loop of the catalytic domain. Kinase-deficient Lck expressed in fibroblasts that do not express any endogenous Lck has been shown to be phosphorylated at Tyr-394 following H(2)O(2) treatment to an extent indistinguishable from that seen with wild type Lck. This finding indicates that a kinase other than Lck itself is capable of phosphorylating Tyr-394. Because fibroblasts express other Src family members, it remained to be determined whether the phosphorylation of Tyr-394 was carried out by another Src family kinase or by an unrelated tyrosine protein kinase. We examined here whether Tyr-394 in kinase-deficient Lck was phosphorylated following exposure of cells devoid of endogenous Src family kinase activity to H(2)O(2). Strikingly, treatment of such cells with H(2)O(2) led to the phosphorylation of Tyr-394 to an extent identical to that seen with wild type Lck, demonstrating that Src family kinases are not required for H(2)O(2)-induced phosphorylation of Lck. Furthermore, this efficient phosphorylation of Lck at Tyr-394 in non-lymphoid cells suggests the existence of an ubiquitous activator of Src family kinases.     

Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.

The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.     

CD45 down-regulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells

The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45(+) T cells and a more sustained, robust tyrosine phosphorylation signal in CD45(-) T cells. This signal resulted in F-actin ring formation and round spreading in the CD45(+) cells and polarized, elongated cell spreading in CD45(-) cells. The enhanced signal in the CD45(-) cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45(+) cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45(-) cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading.     

The noncatalytic domains of Lck regulate its dephosphorylation by CD45

The Src-family tyrosine kinase, Lck, contains two key regulatory phosphotyrosine residues, tyrosine 394 (Tyr-394) and tyrosine 505 (Tyr-505), both of which can be dephosphorylated by CD45. Here, the interaction of CD45 with its substrate, Lck, was determined to be complex, involving multiple interactions with both the catalytic and noncatalytic regions of Lck. CD45 preferentially dephosphorylated Tyr-394 over Tyr-505 in Lck. This was not due to sequence specificity surrounding the phosphotyrosine, but was due to the noncatalytic domains of Lck. The interactions with the noncatalytic domains of Lck and CD45 enhanced the dephosphorylation of Tyr-394 whereas intramolecular interactions within Lck reduced, but did not abolish, the dephosphorylation of Tyr-505. This demonstrates that the noncatalytic domains of Lck regulate the dephosphorylation of both Tyr-394 and Tyr-505 by CD45.     

Lck dephosphorylation at Tyr-394 and inhibition of T cell antigen receptor signaling by Yersinia phosphatase YopH

A key virulence factor for Yersinia pestis, the etiologic agent of plague, is the tyrosine phosphatase YopH, which the bacterium injects into host cells. We report that treatment of human T lymphocytes with a recombinant membrane-permeable YopH resulted in severe reduction in intracellular tyrosine phosphorylation and inhibition of T cell activation. The primary signal transducer for the T cell antigen receptor, the Lck tyrosine kinase, was specifically precipitated by a substrate-trapping YopH mutant, and Lck was dephosphorylated at its positive regulatory site, Tyr-394, in cells containing active YopH. By turning off Lck, YopH blocks T cell antigen receptor signaling at its very first step, effectively preventing the development of a protective immune response against this lethal bacterium.     

Divergent roles of SHP-2 in ERK activation by leptin receptors

The protein tyrosine phosphatase SHP-2 has been proposed to serve as a regulator of leptin signaling, but its specific roles are not fully examined. To directly investigate the role of SHP-2, we employed dominant negative strategies in transfected cells. We show that a catalytically inactive mutant of SHP-2 blocks leptin-stimulated ERK phosphorylation by the long leptin receptor, ObRb. SHP-2, lacking two C-terminal tyrosine residues, partially inhibits ERK phosphorylation. We find similar effects of the SHP-2 mutants after examining stimulation of an ERK-dependent egr-1 promoter-construct by leptin. We also demonstrate ERK phosphorylation and egr-1 mRNA expression in the hypothalamus by leptin. Analysis of signaling by ObRb lacking intracellular tyrosine residues or by the short leptin receptor, ObRa, enabled us to conclude that two pathways are critical for ERK activation. One pathway does not require the intracellular domain of ObRb, whereas the other pathway requires tyrosine residue 985 of ObRb. The phosphatase activity of SHP-2 is required for both pathways, whereas activation of ERK via Tyr-985 of ObRb also requires tyrosine phosphorylation of SHP-2. SHP-2 is thus a positive regulator of ERK by leptin receptors, and both the adaptor function and the phosphatase activity of SHP-2 are critical for this regulation.     

Tyrosine kinase, p56lck-induced cell motility, and urokinase-type plasminogen activator secretion involve activation of epidermal growth factor receptor/extracellular signal regulated kinase pathways

We have recently reported that tyrosine kinase, p56(lck) regulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through tyrosine phosphorylation of IkappaBalpha following hypoxia/reoxygenation (Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 52598-52612). However, the role of hypoxia/reoxygenation (H/R) on ERK1/2-mediated uPA secretion and cell motility and the involvement of p56(lck) and EGF receptor in these processes in breast cancer cells is not well defined. We provide here evidence that H/R induces Lck kinase activity and Lck-dependent tyrosine phosphorylation of EGF receptor in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. H/R also stimulates MEK-1 and ERK1/2 phosphorylations, and H/R-induced phosphorylations were suppressed by the dominant negative form of Lck (DN Lck, K273R) as well as pharmacological inhibitors of EGF receptor and Lck indicating that EGF receptors and Lck are involved in these processes. Transfection of these cells with wild type Lck or Lck F505 (Y505F) but not with Lck F394 (Y394F) induced phosphorylations of EGF receptor followed by MEK-1 and ERK1/2, suggesting that Lck is upstream of EGF receptor and Tyr-394 of Lck is crucial for these processes. H/R also induced uPA secretion and cell motility in these cells. DN Lck and inhibitors of Lck, EGF receptor, and MEK-1 suppressed H/R-induced uPA secretion and cell motility. To our knowledge, this is the first report that p56(lck) in presence of H/R regulates MEK-1-dependent ERK1/2 phosphorylation and uPA secretion through tyrosine phosphorylation of EGF receptor, and it further demonstrates that all of these signaling molecules ultimately control the motility of breast cancer cells.     

SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase phosphorylation and activity.

Ligation of the T cell antigen receptor (TCR) activates the Src family tyrosine kinase p56 Lck, which, in turn, phosphorylates a variety of intracellular substrates. The phosphatidylinositol 3-kinase (PI3K) and the tyrosine phosphatase SHP-1 are two Lck substrates that have been implicated in TCR signaling. In this study, we demonstrate that SHP-1 co-immunoprecipitates with the p85 regulatory subunit of PI3K in Jurkat T cells, and that this association is increased by ligation of the TCR complex. Co-expression of SHP-1 and PI3K with a constitutively activated form of Lck in COS7 cells demonstrated the carboxyl-terminal SH2 domain of PI3K to inducibly associate with the full-length SHP-1 protein. By contrast, a truncated SHP-1 mutant lacking the Lck phosphorylation site (Tyr(564)) failed to bind p85. Wild-type but not catalytically inactive SHP-1 induced dephosphorylation of p85. Furthermore, expression of SHP-1 decreased PI3K enzyme activity in anti-phosphotyrosine immunoprecipitates and phosphorylation of serine 473 in Akt, a process dependent on PI3K activity. These results indicate the presence of a functional interaction between PI3K and SHP-1 and suggest that PI3K signaling, which has been implicated in cell proliferation, apoptosis, cytoskeletal reorganization, and many other biological activities, can be regulated by SHP-1 in T lymphocytes.     

Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8

Efficient expression of innate immunity is critically dependent upon the capacity of the neutrophil to be activated rapidly in the face of an acute threat and to involute once that threat has been eliminated. Here we report a novel mechanism regulating neutrophil survival dynamically through the tyrosine phosphorylation or dephosphorylation of caspase-8. Caspase-8 is tyrosine-phosphorylated in freshly isolated neutrophils but spontaneously dephosphorylates in culture, in association with the progression of constitutive apoptosis. Phosphorylation of caspase-8 on Tyr-310 facilitates its interaction with the Src-homology domain 2 containing tyrosine phosphatase-1 (SHP-1) and enables SHP-1 to dephosphorylate caspase-8, permitting apoptosis to proceed. The non-receptor tyrosine kinase, Lyn, can phosphorylate caspase-8 on Tyr-397 and Tyr-465, rendering it resistant to activational cleavage and inhibiting apoptosis. Exposure to lipopolysaccharide reduces SHP-1 activity and binding to caspase-8, caspase-8 activity, and rates of spontaneous apoptosis. SHP-1 activity is reduced and Lyn increased in neutrophils from patients with sepsis, in association with profoundly delayed apoptosis; inhibition of Lyn can partially reverse this delay. Thus the phosphorylation and dephosphorylation of caspase-8, mediated by Lyn and SHP-1, respectively, represents a novel, dynamic post-translational mechanism for the regulation of neutrophil apoptosis whose dysregulation contributes to persistent neutrophil survival in sepsis.     

Integrins enhance platelet-derived growth factor (PDGF)-dependent responses by altering the signal relay enzymes that are recruited to the PDGF beta receptor.

Since the extracellular matrix (ECM) can promote platelet-derived growth factor (PDGF)-dependent responses, we hypothesized that the ECM mediates this effect by preventing the PDGF beta receptor (betaPDGFR) from associating with the negative regulator, RasGAP (the GTPase-activating protein of Ras). We found that binding of RasGAP to the wild-type betaPDGFR was decreased; the activation of Ras and Erk was enhanced, and [3H]thymidine uptake was better in cells cultured on fibronectin than in cells cultured on polylysine. To investigate the mechanism by which culturing cells on fibronectin diminished the recruitment of RasGAP to the betaPDGFR, we focused on SHP-2 since it dephosphorylates the betaPDGFR at the phosphotyrosine required for binding of RasGAP. Culturing cells on fibronectin increased the amount of SHP-2 that associated with the betaPDGFR. Furthermore, cells expressing receptor mutants that failed to associate with SHP-2 were insensitive to fibronectin. The ECM enhances PDGF-dependent responses by increasing the association of SHP-2 with the betaPDGFR, which in turn decreases the time that RasGAP interacts with the receptor. Thus, fibronectin changes PDGF-dependent signaling and biological responses by altering the signal relay enzymes that are recruited to the receptor.     

Activation of ZAP-70 through specific dephosphorylation at the inhibitory Tyr-292 by the low molecular weight phosphotyrosine phosphatase (LMPTP)

The ZAP-70 protein-tyrosine kinase plays a central role in signaling from the T cell antigen receptor. Recruitment and activation of ZAP-70 are transient and are terminated by phosphorylation of negative regulatory tyrosine residues and dephosphorylation of positively acting sites. We report that the low molecular weight protein-tyrosine phosphatase (LMPTP) specifically dephosphorylates the negative regulatory Tyr-292 of ZAP-70, thereby counteracting inactivation of ZAP-70. Expression of low levels of LMPTP resulted in increased ZAP-70 phosphorylation, presumably at the activating Tyr-493 and other sites, increased kinase activity, and augmented downstream signaling to the mitogen-activated protein kinase pathway. The ZAP-70 Y292F mutant was not affected by LMPTP. Our results indicate that LMPTP, like CD45, dephosphorylates a negative regulatory tyrosine site in a protein-tyrosine kinase and thereby strengthens T cell receptor signaling.     

Identification of substrates of human protein-tyrosine phosphatase PTPN22

Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3epsilon, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) zeta was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRzeta, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRzeta in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRzeta when both were coexpressed in 293T cells, establishing TCRzeta as a direct substrate of PTPN22.     

Differential regulation of components of the focal adhesion complex by heregulin: role of phosphatase SHP-2.

Heregulin (HRG) has been implicated in the progression of breast cancer cells to a malignant phenotype, a process that involves changes in cell motility and adhesion. Here we demonstrate that HRG differentially regulates the site-specific phosphorylation of the focal adhesion components focal adhesion kinase (FAK) and paxilin in a dose-dependent manner. HRG at suboptimal doses (0.01 and 0.1 nM) increased adhesion of cells to the substratum, induced phosphorylation of FAK at Tyr-577, -925, and induced formation of well-defined focal points in breast cancer cell line MCF-7. HRG at a dose of 1 nM, increased migratory potential of breast cancer cells, selectively dephosphorylated FAK at Tyr-577, -925, and paxillin at Tyr-31. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by HRG stimulation. FAK associated with HER2 only in response to 0.01 nM HRG. In contrast, 1 nM HRG induced activation and increased association of tyrosine phosphatase SHP-2 with HER2 but decreased association of HER2 with FAK. Expression of dominant-negative SHP-2 blocked HRG-mediated dephosphorylation of FAK and paxillin, leading to persistent accumulation of mature focal points. Our results suggest that HRG differentially regulates signaling from focal adhesion complexes through selective phosphorylation and dephosphorylation and that tyrosine phosphatase SHP-2 has a role in the HRG signaling.     

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.     

Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.     

Cutting edge: differential segregation of the SRC homology 2-containing protein tyrosine phosphatase-1 within the early NK cell immune synapse distinguishes noncytolytic from cytolytic interactions

Inhibitory NK receptors with ligand specificity for MHC class I recruit Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) phosphatase and prevent autocytotoxicity. Activation of SHP-1 depends upon Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of the inhibitory receptor. In this study we demonstrate, by quantitative temporal analysis, that talin, Lck, and SHP-1 are recruited to the synapse within 1 min in both cytolytic and noncytolytic conjugates. Polarization of talin and Lck rapidly disappears in the noncytolytic interactions but persists in cytolytic interactions, where protein kinase C-theta;, Src homology 2 domain-containing leukocyte protein of 76 kDa, and lysosomes are recruited within 5 min. At 1 min SHP-1 clusters in the periphery of the cytolytic synapse, whereas it clusters in the center of the noncytolytic synapse. Lck has multifocal distribution in both synapses consistent with the shared requirement for early tyrosine phosphorylation. Our studies indicate that the spatial location of SHP-1 in the synapse distinguishes noncytolytic from cytolytic interactions within the first minute.     

Phosphorylation of SHP-2 regulates interactions between the endoplasmic reticulum and focal adhesions to restrict interleukin-1-induced Ca2+ signaling

Interleukin-1 (IL-1)-induced Ca2+ signaling in fibroblasts is constrained by focal adhesions. This process involves the proteintyrosine phosphatase SHP-2, which is critical for IL-1-induced phosphorylation of phospholipase Cgamma1, thereby enhancing IL-1-induced Ca2+ release and ERK activation. Currently, the mechanisms by which SHP-2 modulates Ca2+ release from the endoplasmic reticulum are not defined. We used immunoprecipitation and fluorescence protein-tagged SHP-2 or endoplasmic reticulum (ER)-protein expression vectors, and an ER-specific calcium indicator, to examine the functional relationships between SHP-2, focal adhesions, and IL-1-induced Ca2+ release from the ER. By total internal reflection fluorescence microscopy to image subplasma membrane compartments, SHP-2 co-localized with the ER-associated proteins calnexin and calreticulin at sites of focal adhesion formation in fibroblasts. IL-1beta promoted time-dependent recruitment of SHP-2 and ER proteins to focal adhesions; this process was blocked in cells treated with small interfering RNA for SHP-2 and in cells expressing a Y542F SHP-2 mutant. IL-1 stimulated inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the ER subjacent to the plasma membrane that was tightly localized around fibronectin-coated beads and was reduced 4-fold in cells expressing Tyr-542 SHP-2 mutant. In subcellular fractions enriched for ER proteins, immunoprecipitation demonstrated that IL-1-enhanced association of SHP-2 with the type 1 inositol 1,4,5-trisphosphate receptor was dependent on Tyr-542 of SHP-2. We conclude that Tyr-542 of SHP-2 modulates IL-1-induced Ca2+ signals and association of the ER with focal adhesions.     

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.     

EGF-stimulation activates the nuclear localization signal of SHP-1

Protein tyrosine phosphatase SHP-1 plays a critical role in the regulation of a variety of intracellular signaling pathways. SHP-1 is predominantly expressed in the cells of hematopoietic origin, and is recognized as a negative regulator of lymphocyte development and activation. SHP-1 consists of two Src homology 2 (SH2) domains and one protein tyrosine phosphatase (PTP) domain followed by a highly basic C-terminal tail containing tyrosyl phosphorylation sites. It is unclear how the C-terminal tail regulates SHP-1 function. We report the examination of the subcellular localization of a variety of truncated or mutated SHP-1 proteins fused with enhanced green fluorescent protein (EGFP) protein at either the N-terminal or the C-terminal end in different cell lines. Our data demonstrate that a nuclear localization signal (NLS) is located in the C-terminal tail of SHP-1 and the signal is primarily defined by three amino-acid residues (KRK) at the C-terminus. This signal is generally blocked in the native protein and can be exposed by fusing EGFP at the appropriate position or by domain truncation. We have also revealed that this NLS of SHP-1 is triggered by epidermal growth factor (EGF) stimulation and mediates translocation of SHP-1 from the cytosol to the nucleus in COS7 cell lines. These results not only demonstrate the importance of the C-terminal tail of SHP-1 in the regulation of nuclear localization, but also provide insights into its role in SHP-1-involved signal transduction pathways.