Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

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Involvement of the SH3 domain in Ca2+-mediated regulation of Src family kinases

Biochimie ◽

10.1016/j.biochi.2006.01.013 ◽

2006 ◽

Vol 88 (7) ◽

pp. 905-911 ◽

Cited By ~ 3

Author(s):

A.N.A. Monteiro

Keyword(s):

Sh3 Domain ◽

Src Family Kinases

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Interaction between the SH3 domain of Src family kinases and the proline-rich motif of HTLV-1 p13: a novel mechanism underlying delivery of Src family kinases to mitochondria

Biochemical Journal ◽

10.1042/bj20101650 ◽

2011 ◽

Vol 439 (3) ◽

pp. 505-518 ◽

Cited By ~ 17

Author(s):

Elena Tibaldi ◽

Andrea Venerando ◽

Francesca Zonta ◽

Carlo Bidoia ◽

Elisa Magrin ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Sh3 Domain ◽

Src Family Kinases ◽

General Mechanism ◽

Cell Leukaemia ◽

Mitochondrial Localization ◽

Leukaemia Virus ◽

Human T Cell ◽

Mitochondrial Functions ◽

Localization Signal

The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type 1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions.

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SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding

10.1101/2022.01.13.475555 ◽

2022 ◽

Author(s):

Ben F Brian ◽

Frances V Sjaastad ◽

Tanya S Freedman

Keyword(s):

Ubiquitin Ligase ◽

Kinase Activity ◽

Immune Cell ◽

Sh3 Domain ◽

Src Family Kinases ◽

Negative Regulator ◽

Regulatory Function ◽

Membrane Microdomains ◽

Activation Loop ◽

Human Polymorphism

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, i.e., Lck, Fyn, Lyn, Hck, Fgr, Blk, Src, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that nucleates an autoinhibitory complex. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (Pep/LYP), which dephosphorylate the SFK activation loop to promote autoinhibition. High local concentrations of Csk are required to promote its negative-regulatory function, and Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains. Purified Csk also homodimerizes in solution through an interface that overlaps the phosphatase binding site. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Csk W47A, an SH3-domain mutant commonly used to block PTPN22 binding, also impairs homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with both Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

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Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095017 ◽

1972 ◽

Vol 30 ◽

pp. 350-351

Author(s):

K. Shankar Narayan ◽

Kailash C. Gupta ◽

Tohru Okigaki

Keyword(s):

Ultraviolet Light ◽

Mammalian Cells ◽

Biological Effects ◽

Survival Rates ◽

Chinese Hamster ◽

Cell Types ◽

Differential Sensitivity ◽

Ultrastructural Changes ◽

Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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