Phosphorylation of the ras GTPase-activating protein (GAP) by the p93c-fes protein-tyrosine kinase in vitro and formation of GAP-fes complexes via an SH2 domain-dependent mechanism

Biochemistry ◽  
1993 ◽  
Vol 32 (39) ◽  
pp. 10519-10525 ◽  
Author(s):  
Scott J. Hjermstad ◽  
Scott D. Briggs ◽  
Thomas E. Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Sh2 Domain ◽  
Gtpase Activating Protein ◽  
Protein Tyrosine ◽  
Ras Gtpase ◽  
Dependent Mechanism

scholarly journals The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src.

1995 ◽  
Vol 15 (9) ◽  
pp. 4908-4920 ◽  
Author(s):  
K Neet ◽  
T Hunter
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Sh2 Domain ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Protein Tyrosine ◽  
Src Activation ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.

scholarly journals Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the FcεRI Pathway in Mast Cells

2008 ◽  
Vol 29 (2) ◽  
pp. 389-401 ◽  
Author(s):  
Victor A. McPherson ◽  
Stephanie Everingham ◽  
Robert Karisch ◽  
Julie A. Smith ◽  
Christian M. Udell ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Regulatory Protein ◽  
Sh2 Domain ◽  
Wild Type ◽  
Protein Tyrosine ◽  
Bar Domain ◽  
Sh2 Domains

ABSTRACT This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcεRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcεRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcεRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcεRI signaling and potential regulation the actin reorganization in mast cells.

Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase

1993 ◽  
Vol 13 (12) ◽  
pp. 7708-7717
Author(s):  
K V Prasad ◽  
R Kapeller ◽  
O Janssen ◽  
H Repke ◽  
J S Duke-Cohan ◽  
...  
Keyword(s):  
T Cells ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Activation Process ◽  
Lipid Kinase ◽  
Sh3 Domains ◽  
Protein Tyrosine ◽  
Hiv 1

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

Characterization of elk, a brain-specific receptor tyrosine kinase

1991 ◽  
Vol 11 (5) ◽  
pp. 2496-2502
Author(s):  
V Lhoták ◽  
P Greer ◽  
K Letwin ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Catalytic Domain ◽  
Signal Sequence ◽  
Tyrosine Kinase Domain ◽  
Rat Tissues ◽  
Protein Tyrosine

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.

scholarly journals Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Matthias Kästle ◽  
Camilla Merten ◽  
Roland Hartig ◽  
Thilo Kaehne ◽  
Ardiyanto Liaunardy-Jopeace ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Enzymatic Activity ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Jurkat T Cells

Abstract Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

scholarly journals The Human c-Fes Tyrosine Kinase Binds Tubulin and Microtubules through Separate Domains and Promotes Microtubule Assembly

2004 ◽  
Vol 24 (21) ◽  
pp. 9351-9358 ◽  
Author(s):  
Charles E. Laurent ◽  
Frank J. Delfino ◽  
Haiyun Y. Cheng ◽  
Thomas E. Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Morphological Changes ◽  
Neuronal Cells ◽  
Coiled Coil ◽  
Cell Types ◽  
Sh2 Domain ◽  
Microtubule Assembly

ABSTRACT The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.

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