The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

Download Full-text

Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4648-4656.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4648-4656

Author(s):

R B Birge ◽

J E Fajardo ◽

C Reichman ◽

S E Shoelson ◽

Z Songyang ◽

...

Keyword(s):

Fusion Protein ◽

Tyrosine Phosphorylation ◽

Cell Transformation ◽

Tyrosine Phosphatase ◽

State Level ◽

Sh2 Domain ◽

Sequence Specificity ◽

Src Homology

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

Download Full-text

Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4648 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4648-4656 ◽

Cited By ~ 166

Author(s):

R B Birge ◽

J E Fajardo ◽

C Reichman ◽

S E Shoelson ◽

Z Songyang ◽

...

Keyword(s):

Fusion Protein ◽

Tyrosine Phosphorylation ◽

Cell Transformation ◽

Tyrosine Phosphatase ◽

State Level ◽

Sh2 Domain ◽

Sequence Specificity ◽

Src Homology

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

Download Full-text

Expression and catalytic activity of the tyrosine phosphatase PTP1C is severely impaired in motheaten and viable motheaten mice.

Journal of Experimental Medicine ◽

10.1084/jem.178.6.2157 ◽

1993 ◽

Vol 178 (6) ◽

pp. 2157-2163 ◽

Cited By ~ 168

Author(s):

M Kozlowski ◽

I Mlinaric-Rascan ◽

G S Feng ◽

R Shen ◽

T Pawson ◽

...

Keyword(s):

Tyrosine Phosphatase ◽

Critical Role ◽

Sh2 Domain ◽

Loss Of Function ◽

Phosphotyrosine Phosphatase ◽

Gene Encoding ◽

Phosphatase Domain ◽

Sh2 Domains ◽

Src Homology

Mutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a COOH-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (mev) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the mev mutation creates an insertion or deletion in the phosphatase domain. No PTP1C RNA or protein could be detected in the hemopoietic tissues of me mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe mev mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of mev PTP1C bound normally to phosphotyrosine-containing ligands in vitro. Nevertheless, the mev mutation induced a marked reduction in PTP1C activity. These observations provide strong evidence that the motheaten phenotypic results from loss-of-function mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.

Download Full-text

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Molecular and Cellular Biology ◽

10.1128/mcb.01202-09 ◽

2010 ◽

Vol 30 (12) ◽

pp. 2850-2861 ◽

Cited By ~ 14

Author(s):

Andrei M. Vacaru ◽

Jeroen den Hertog

Keyword(s):

Protein Tyrosine Phosphatase ◽

Protein Tyrosine Kinase ◽

Tyrosine Phosphatase ◽

Sh2 Domain ◽

Serine Phosphorylation ◽

Receptor Protein ◽

Protein Tyrosine ◽

Receptor Protein Tyrosine Phosphatase ◽

Src Homology ◽

Nih 3T3

ABSTRACT Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.

Download Full-text

Tyrosine Phosphorylation of Selected Secretory Carrier Membrane Proteins, SCAMP1 and SCAMP3, and Association with the EGF Receptor

Molecular Biology of the Cell ◽

10.1091/mbc.9.7.1661 ◽

1998 ◽

Vol 9 (7) ◽

pp. 1661-1674 ◽

Cited By ~ 22

Author(s):

Theodore T. Wu ◽

J. David Castle

Keyword(s):

Membrane Proteins ◽

Egf Receptor ◽

Tyrosine Phosphatase ◽

Chinese Hamster ◽

Tyrosine Residue ◽

Cell Interior ◽

Murine Fibroblasts ◽

Secretory Carrier Membrane Proteins

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

Download Full-text

Synthesis and in Vitro Evaluation of a Peptidomimetic Inhibitor Targeting the Src Homology 2 (SH2) Domain of STAT6

ACS Medicinal Chemistry Letters ◽

10.1021/ml4003919 ◽

2013 ◽

Vol 5 (1) ◽

pp. 69-72 ◽

Cited By ~ 15

Author(s):

Pietro Morlacchi ◽

Pijus K. Mandal ◽

John S. McMurray

Keyword(s):

Sh2 Domain ◽

In Vitro Evaluation ◽

Src Homology 2 ◽

Src Homology

Download Full-text

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.509-517.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 509-517

Author(s):

W Li ◽

R Nishimura ◽

A Kashishian ◽

A G Batzer ◽

W J Kim ◽

...

Keyword(s):

Growth Factor ◽

Intracellular Signaling ◽

Egf Receptor ◽

Tyrosine Phosphatase ◽

Growth Factor Receptors ◽

Ras Signaling ◽

Pdgf Receptor ◽

Phosphotyrosine Phosphatase ◽

Src Homology

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.

Download Full-text

Triazolopyrimidinium salts: discovery of a new class of agents for cancer therapy

Future Medicinal Chemistry ◽

10.4155/fmc-2019-0317 ◽

2020 ◽

Vol 12 (5) ◽

pp. 387-402

Author(s):

Mariateresa Badolato ◽

Fabrizio Manetti ◽

Antonio Garofalo ◽

Francesca Aiello

Keyword(s):

Anticancer Agents ◽

Docking Studies ◽

In Vitro Models ◽

Sh2 Domain ◽

Activity Profile ◽

Cancer Cell Growth ◽

New Class ◽

Src Homology 2 ◽

Src Homology

Aim: The [1,2,4]triazolo[1,5- a]pyrimidine core is highly privileged in medicinal chemistry due to its versatile pharmacological activity profile. Recently, the search for novel anticancer agents has focused on [1,2,4]triazolo[1,5- a]pyrimidine derivatives. Results: Our hit functionalization has led to the discovery of new [1,2,4]triazolo[1,5- a]pyrimidinium salts with potential anticancer activity. Among a small library of molecules, compound 9 significantly inhibits cancer cell growth in a panel of in vitro models. Molecular docking studies and preliminary binding assay have displayed that 9 could directly bind the Src homology 2 (SH2) domain of STAT3 protein. Conclusion: Compound 9 is a novel promising lead compound that motivates additional evaluation of [1,2,4]triazolo[1,5- a]pyrimidinium salts as novel potential chemotherapeutics.

Download Full-text

A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2)

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011422 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3563-3575 ◽

Cited By ~ 2

Author(s):

Zachary Hartman ◽

Werner J. Geldenhuys ◽

Yehenew M. Agazie

Keyword(s):

Amino Acid ◽

Active Site ◽

Specific Binding ◽

Tyrosine Phosphatase ◽

Strong Inhibitor ◽

Selective Binding ◽

Specific Amino Acid ◽

Src Homology ◽

Her2 Signaling

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1–4 or the human homologs HER1–4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

Download Full-text