Epidermal growth factor stimulates the tyrosine phosphorylation of SHPS-1 and association of SHPS-1 with SHP-2, a SH2 domain-containing protein tyrosine phosphatase

SHPS-1 is a 120 kDa glycosylated receptor-like protein that contains immunoglobulin-like domains in its extracellular region and four potential tyrosine phosphorylation for SH2 domain binding sites in its cytoplasmic region. Epidermal growth factor (EGF) stimulated the rapid tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2, a protein tyrosine phosphatase containing SH2 domains, in Chinese hamster ovary cells overexpressing human EGF receptors. In the cells overexpressing SHPS-1, the tyrosine phosphorylation of SHPS-1 was more evident than that observed in parent cells. However, overexpression of SHPS-1 alone did not affect the activation of MAP kinase in response to EGF. These results suggest that SHPS-1 may be involved in the recruitment of SHP-2 from the cytosol to the plasma membrane in response to EGF.     

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.     

The protein tyrosine phosphatase SHP-1 regulates integrin-mediated adhesion of macrophages

The Src homology 2 domain phosphatase-1 (SHP-1) is a tyrosine phosphatase containing two amino-terminal SH2 domains and is expressed primarily by hematopoietic-derived cells [1]. The viable motheaten (Hcphme-v) mutant mice (mev) suffer from progressive inflammation due to a deficiency of SHP-1 enzyme activity [2,3] and die at 3-4 months of age from macrophage and neutrophil accumulation in the lung [4]. The mechanism by which SHP-1 deficiency leads to inflammation is unknown. We found that macrophages from mev mice adhered and spread to a greater extent than normal macrophages through alpha m beta 2 integrin-mediated contacts. Whereas macrophages deficient in the transmembrane tyrosine phosphatase CD45 (CD45-/-) spontaneously detached from alpha m beta 2 integrin contacts [5], cells deficient in both CD45 and SHP-1 did not. In SHP-1 deficient macrophages there was a 10-15-fold increase in D-3 phospholipid products of phosphatidylinositol (PI) 3-kinase. Concomitantly, there was a 2-5-fold increase in membrane-associated PI 3-kinase activity in mev macrophages relative to normal macrophages. Treatment of macrophages with the PI 3-kinase inhibitors wortmannin or LY294002 resulted in a dramatic detachment of cells, indicating that PI 3-kinase activity is required for adhesion. These data demonstrate that SHP-1 is necessary for detachment from alpha m beta 2 integrin-mediated contacts in primary macrophages and suggest that a defect in this pathway may contribute to inflammatory disease.     

Reversible regulation of SHP-1 tyrosine phosphatase activity by oxidation

BACKGROUND AND PURPOSE: Randomized clinical trials testing aspirin in relatively low-risk, middle-aged people have consistently shown small increases in stroke associated with aspirin use. We analyzed the relationship between the regular use of aspirin and incident ischemic and hemorrhagic stroke among people aged 65 years or older participating in the Cardiovascular Health Study. METHODS: We conducted a multivariate analysis of incident stroke rates in a prospectively assessed, observational cohort of 5011 elderly people followed for a mean of 4.2 years. RESULTS: Participants had a mean age of 72 years, and 58% were women. Twenty-three percent used aspirin frequently, and 17% used aspirin infrequently at study entry. Frequent aspirin use was associated with an increased rate of ischemic stroke compared with nonusers (relative risk= 1.6; 95% confidence interval [CI], 1.2 to 2.2; P=0.001). After adjustment for other stroke risk factors, women who used aspirin frequently or infrequently at study entry had a 1.8-fold (95% CI, 1.2 to 2.8) and 1.6-fold (95% CI, 0.9 to 3.0) increased risk of ischemic stroke, respectively (P<0.01, test for trend), compared with nonusers. In men, aspirin use was not statistically significantly associated with stroke risk. Findings were similar when aspirin use in the years before the incident stroke was used in the modeling. Aspirin use at entry was also associated with a 4-fold (95% CI, 1.6 to 10.0) increase in risk of hemorrhagic stroke for both infrequent and frequent users of aspirin (P=0.003). CONCLUSIONS: Aspirin use was associated with increased risks of ischemic stroke in women and hemorrhagic stroke overall in this elderly cohort, after adjustment for other stroke predictors. The possibility exists of confounding by reasons for aspirin use rather than cause and effect. Whether regular aspirin use increases stroke risk for elderly people without cardiovascular disease can only be determined by randomized clinical trials.     

Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine dephosphorylation and inhibition of mitogen-activated protein kinase in human mononuclear phagocytes. Role of the Src homology 2 containing tyrosine phosphatase 1

Lipoarabinomannan (LAM) is a putative virulence factor of Mycobacterium tuberculosis that inhibits monocyte functions, and this may involve antagonism of cell signaling pathways. The effects of LAM on protein tyrosine phosphorylation in cells of the human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein kinase. To examine whether these effects of LAM could be related to activation of a phosphatase, fractions from LAM-treated cells were analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells, using phosphorylated mitogen-activated protein kinase as substrate, indicated that LAM promoted increased activity of SHP-1 in vivo. LAM also activated SHP-1 directly in vitro. Exposure of cells to LAM also attenuated the expression of tumor necrosis factor-alpha, interleukin-12, and major histocompatibility class II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.     

The B cell surface protein CD72 recruits the tyrosine phosphatase SHP-1 upon tyrosine phosphorylation

Activation signals of lymphocytes are negatively regulated by the membrane molecules carrying the immunoreceptor tyrosine-based inhibition motif (ITIM). Upon tyrosine phosphorylation, ITIMs recruit SH2-containing phosphatases such as SHP-1, resulting in down-modulation of cell activation. We showed that the cytoplasmic domain of the CD72 molecule carries an ITIM and is associated in vitro with SHP-1 upon tyrosine phosphorylation. Moreover, cross-linking of B cell Ag receptor (BCR) enhances both tyrosine phosphorylation of CD72 and association of CD72 with SHP-1 in B cell line WEHI-231. These results indicate that CD72 recruits SHP-1 upon tyrosine phosphorylation induced by BCR signaling, suggesting that CD72 is a negative regulator of BCR signaling.     

Multiple phosphorylation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B by casein kinase II and p60c-src in vitro

The objectives of this study were to evaluate milk choline as an indicator of choline absorption and to use milk choline to evaluate the efficacy of a rumen-protected choline supplement. In a preliminary 4-wk experiment, two Holstein cows in early lactation were abomasally infused with either 0 or 60 g/d of choline chloride in 2 L of water, which was used as a carrier. Choline infusion increased milk choline secretion from 1.95 to 3.95 g/d during the 2-wk choline infusion period. In Experiment 2, four Holstein cows in early lactation were abomasally infused with 0, 25, 50, and 75 g/d of choline chloride in 2 L of water using a 4 x 4 Latin square design with 1-wk experimental periods. Milk choline secretion was 2.56, 3.62, 3.72, and 3.82 g/d for the respective choline treatments. In Experiment 3, 10 Holstein cows in midlactation were fed either 0 or 50 g/d of choline using an experimental rumen-protected choline supplement during a 2-wk experiment. Milk choline secretion was increased from 2.12 to 2.99 g/d with the supplemental choline. Results of these experiments demonstrated that milk choline is responsive to postruminal choline supply and can be used as a qualitative indicator of choline absorption.     

Regulation of Bcr-Abl-induced SAP kinase activity and transformation by the SHPTP1 protein tyrosine phosphatase

The oncogenic Bcr-Abl variant of the c-Abl tyrosine kinase transforms cells by a mechanism dependent on activation of the stress-activated protein kinase (SAPK). Other work has shown that c-Abl interacts with the SHPTP1 protein tyrosine phosphatase in induction of SAPK activity by genotoxic stress. The present studies demonstrate that Bcr-Abl binds constitutively to SHPTP1. We show that Bcr-Abl phosphorylates SHPTP1 on C-terminal Y536 and Y564 sites. The functional significance of the Bcr-Abl/SHPTP1 interaction is supported by the finding that SHPTP1 regulates Bcr-Abl-induced SAPK activity. Importantly, SHPTP1 also decreases Bcr-Abl-dependent transformation of fibroblasts. These findings indicate that SHPTP1 functions as a tumor suppressor in cells transformed by Bcr-Abl.     

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1

BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.     

Purification and cloning of PZR, a binding protein and putative physiological substrate of tyrosine phosphatase SHP-2

Overexpression of a catalytically inactive mutant of tyrosine phosphatase SHP-2 in 293 cells resulted in hyperphosphorylation of a glycoprotein specifically associated with the enzyme. The protein has been purified to near homogeneity. Based on the amino acid sequences of peptides obtained from the protein, a full-length cDNA was isolated. The cDNA encodes a protein with a single transmembrane segment and a signal sequence. The extracellular portion of the protein contains a single immunoglobulin-like domain displaying 46% sequence identity to that of myelin P0, a major structural protein of peripheral myelin. The intracellular segment of the protein shows no significant sequence identity to any known protein except for two immunoreceptor tyrosine-based inhibitory motifs. We name the protein PZR for protein zero related. Transfection of the PZR cDNA in Jurkat cells gave rise to a protein of expected molecular size. Stimulation of cells with pervanadate resulted in tyrosine phosphorylation of PZR and a near-stoichiometric association of PZR with SHP-2. Northern blotting analyses revealed that PZR is widely expressed in human tissues and is particularly abundant in heart, placenta, kidney, and pancreas. As a binding protein and a putative substrate of SHP-2, PZR protein may have an important role in cell signaling.     

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.     

Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.     

Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.     

The protein tyrosine phosphatase SHP-2 is expressed in glial and neuronal progenitor cells, postmitotic neurons and reactive astrocytes

In this study we examined the distribution and developmental profile of the src homology 2 (SH2) domain-containing protein tyrosine phosphatase SHP-2 in the mouse brain. We found that SHP-2 is present in both mitotically active and postmitotic cells in the forebrains of embryonic day 12 (E12) mice. In a developmental study extending from embryonic day 12 to adulthood, Western blotting analysis demonstrated equivalent levels of SHP-2 protein at all of the ages examined. Expression of SHP-2 paralleled the level of enzymatic activity at the different developmental periods. In the adult brain SHP-2 was restricted to diverse classes of neurons, while the majority of glial cells did not express detectable levels of protein. However, reactive astrocytes in response to an ischemic brain injury showed SHP-2 immunolabelling. Our data suggest that SHP-2 may play a role in pathways of neuronal and glial progenitor cells, in a broad spectrum of neuronal responses in the adult brain and in the gliotic response to the injury.     

Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha

Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase casein kinase 2 (CK2) bound to PP2A in vitro and in mitogen-starved cells; binding required the integrity of a sequence motif common to CK2alpha and SV40 small t antigen. Overexpression of CK2alpha resulted in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2alpha inhibited the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2alpha may regulate the deactivation of the mitogen-activated protein kinase pathway.     

Cloning and expression of the chicken protein tyrosine phosphatase SH-PTP2

SH-PTP2 is a protein tyrosine phosphatase which contains two src homology 2 (SH2) domains. A partial cDNA clone encoding chicken SH-PTP2 was generated by RT-PCR and used as a probe to screen several chicken cDNA libraries. Two overlapping cDNA clones were identified and the nucleotide sequence of chicken SH-PTP2 containing the entire protein-coding region was determined. The deduced amino acid sequence shares 98% and 94% identity, respectively with the corresponding human and Xenopus proteins. Northern and Western blot analyses show that chicken SH-PTP2 is expressed ubiquitously like those of mammals and Xenopus. This suggests that chicken SH-PTP2 may have analogous biological roles to those of mammals.     

Regulation of DNA-dependent protein kinase by the Lyn tyrosine kinase

The Src-like protein-tyrosine kinase Lyn is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunits (DNA-PKcs) and Ku DNA-binding components, requires DNA double-stranded breaks for activation. Here we demonstrate that Lyn associates constitutively with DNA-PKcs. The SH3 domain of Lyn interacts directly with DNA-PKcs near a leucine zipper homology domain. We also show that Lyn phosphorylates DNA-PKcs but not Ku in vitro. The interaction between Lyn and DNA-PKcs inhibits DNA-PKcs activity and the ability of DNA-PKcs to form a complex with Ku/DNA. These results support the hypothesis that there are functional interactions between Lyn and DNA-PKcs in the response to DNA damage.