The Src family of protein tyrosine kinases: regulation and functions

Development ◽  
1993 ◽  
Vol 119 (Supplement) ◽  
pp. 57-64
Author(s):  
Sara A. Courtneidge ◽  
Stefano Fumagalli ◽  
Manfred Koegl ◽  
Giulio Superti-Furga ◽  
Geraldine M. Twamley-Stein
Keyword(s):  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Pdgf Receptor ◽  
Intramolecular Association ◽  
Src Family Tyrosine Kinases ◽  
Carboxy Terminal ◽  
Mitogenic Signal

Most of the nine members of the Src family of tyrosine kinases arc restricted in their expression, often to cells of the haematopoietic lineage, while some, particularly Src, Fyn and Yes, are more unbiquitously expressed. We have been studying the functions of Src, Fyn and Yes in fibroblasts. We have shown that stimulation of quiescent fibroblasts with platelet-derived growth factor (PDGF) causes Src, Fyn and Yes to become activated, and to associate transiently with the PDGF receptor. To address the role of Src, Fyn and Yes in the response to PDGF, we have used a dominant negative approach, in which cells were engineered to express catalytically inactive forms of Src kinases. These cells were unable to enter S phase in response to PDGF, and we therefore conclude that Src family tyrosine kinases are required in order for the PDGF receptor to transmit a mitogenic signal. It has previously been shown that the kinase activity of Src is negatively regulated by phosphorylation of tyr 527 in its carboxy-terminal tail. A kinase, Csk, that phosphorylates tyr 527 has recently been identified. We expressed Src in yeast to test the model that phosphorylation of tyr 527 represses activity by promoting intramolecular association between the tail and the SH2 domain. Inducible expression of Src in S. pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in the SH2 domain were as lethal as wild-type Src, but were insensitive to Csk. We interpret these results in favour of an SH2 domain : phosphorylated tail interaction repressing Src activity. However, we have also found that Src molecules containing mutations in the SH3 domain are not regulated by Csk, suggesting that the SH3 domain also functions in the intramolecular regulation of Src activity.

scholarly journals Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins.

1995 ◽  
Vol 15 (12) ◽  
pp. 6829-6837 ◽  
Author(s):  
M Tanaka ◽  
R Gupta ◽  
B J Mayer
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.

scholarly journals Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

1995 ◽  
Vol 15 (1) ◽  
pp. 186-197 ◽  
Author(s):  
S Richard ◽  
D Yu ◽  
K J Blumer ◽  
D Hausladen ◽  
M W Olszowy ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Family Kinases ◽  
Src Family Kinase ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Src Family Tyrosine Kinases

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.

Src induces morphological changes in A431 cells that resemble epidermal differentiation through an SH3- and Ras-independent pathway

1999 ◽  
Vol 112 (17) ◽  
pp. 2913-2924
Author(s):  
F. Jin ◽  
A.B. Reynolds ◽  
M.D. Hines ◽  
P.J. Jensen ◽  
K.R. Johnson ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Morphological Changes ◽  
Sh2 Domain ◽  
Epidermal Differentiation ◽  
A431 Cells ◽  
Src Family Tyrosine Kinases

The role of Src family tyrosine kinases in cellular proliferation is well established; however, their role in cellular differentiation is less well understood. In this study we have investigated the role played by Src in the differentiation of squamous epithelial cells. Transfection of activated Src into A431 cells resulted in morphological changes that resembled epidermal differentiation. When we used Src mutants to characterize the observed phenotypic changes, we found that protein tyrosine kinase activity, correct membrane localization and the activity of the SH2 domain were required, but the SH3 domain was not. Furthermore, downstream activity of Ras was not required for the Src-mediated changes in A431 cells.

scholarly journals Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

1994 ◽  
Vol 14 (5) ◽  
pp. 2883-2894 ◽  
Author(s):  
B J Mayer ◽  
D Baltimore
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Abl Tyrosine Kinase ◽  
Transforming Activity

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3970-3970
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Class Iii ◽  
Primary Resistance

Abstract Abstract 3970 Poster Board III-906 Purpose The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. Results We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation. Conclusion Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

2001 ◽  
Vol 12 (7) ◽  
pp. 2171-2183 ◽  
Author(s):  
Juan Ángel Fresno Vara ◽  
Ma Aurora Domı́nguez Cáceres ◽  
Augusto Silva ◽  
Jorge Martı́n-Pérez
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
Tyrosine Phosphatase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Herbimycin A ◽  
Serine Kinases ◽  
Dependent Cell

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [3H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, andodc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.

scholarly journals The chemotactic response to PDGF-BB: evidence of a role for Ras.

1995 ◽  
Vol 130 (3) ◽  
pp. 725-731 ◽  
Author(s):  
V Kundra ◽  
B Anand-Apte ◽  
L A Feig ◽  
B R Zetter
Keyword(s):  
Chemotactic Response ◽  
Dominant Negative ◽  
Pdgf Receptor ◽  
Gtpase Activating Protein ◽  
Ras Gtpase ◽  
Downstream Effector ◽  
Ras Inhibition ◽  
Receptor Beta ◽  
Constitutively Active

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase-mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF-stimulated chemotaxis but may not be required for fibronectin-stimulated cell motility.

scholarly journals The v-Src SH3 domain binds phosphatidylinositol 3'-kinase.

1993 ◽  
Vol 13 (9) ◽  
pp. 5225-5232 ◽  
Author(s):  
X Liu ◽  
L E Marengere ◽  
C A Koch ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Chicken Embryo ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

Regulatory Role of the Sprouty Gene Family on PDGFβR Fusion Oncogenes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3509-3509
Author(s):  
Silja D. Andradottir ◽  
Magnus K. Magnusson
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Gene Family ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Regulatory Genes ◽  
Dominant Negative ◽  
Regulatory Role ◽  
Thymidine Uptake

Abstract Constitutively activated fusion tyrosine kinases of the Platelet-dervied growth factor β receptor (PDGFβR) family have been described in patients with chronic myelomonocytic leukemia (CMML). Like other tyrosine kinase driven myeloproliferative syndromes, CMML is characterized by progression leading to highly aggressive acute leukemia. In order to understand the progression of these malignancies we are studying tyrosine kinase pathway regulatory genes. In this study we focus on the functional role of the sprouty gene family in the regulation of PDGFβR fusion oncogenes. Sprouty (Spry) has recently been identified as a repressor of receptor tyrosine kinases signaling in vertebrates and invertebrates. The studies of sprouty in the mammalian system have thus far mostly focused on the regulation of the epidermal and fibroblast growth factor receptor, while nothing is known about the possible regulation of PDGF receptors by sprouty proteins and nothing is known about regulation of mutationally activated tyrosine kinases. Expression plasmids containing human sprouty wildtype genes (Spry1-3 WT) were constructed, along with a series of plasmids containing dominant negative variants by site-direct mutagenesis in critically conserved domains [Spry1(Y53F), Spry2(Y55F), Spry3(Y27F)]. Stable cell lines containing these plasmids have been generated in the BaF3 background with or without the constitutively activated Rabaptin-5/PDGFβR (R/P) fusion oncoprotein. Effects on cell growth and downstream signaling events were studied. Spry1 WT and Spry3 WT signifcantly inhibit growth of R/P transformed BaF3 cell lines. This inhibition was much more pronounced in IL3 depleted media indicating that the inhibition is mediated through PDGFβR tyrosine kinase inhibition. The dominant negative forms, Spry1(Y53F) and Spry3(Y27F) stimulated growth of the the same BaF3 cell lines. Results from [3H]thymidine uptake studies in these cell lines showed decreased uptake in Spry1 WT and Spry3 WT transduced cells and increased uptake in the dominant negative forms, indicating that the effects are through increased proliferation rather than decreased apoptosis. Interestingly, R/P transformed BaF3 cell lines transfected with plasmid containing Spry2 WT and Spry2(Y55F) showed inverse results, Spry2 WT stimulated growth while Spry2(Y55F) inhibited growth. A possible explanation for stimulatory effects of Spry2 is that this Spry variant contains a Cbl binding domain previously shown to prevent Cbl mediated ubiquitylation and degradation of RTKs. We are currently studying the downstream targets of the Spry regulation of PDGFβR focusing on Ras and MAPkinase pathways. In conclusion, we have shown that Spry1 and Spry3 inhibits growth of PDGFβR transformed BaF3 cell lines, while Spry2 stimulates growth. This is the first evidence for regulatory role of Sprouty genes in activated fusion tyrosine kinase. This conserved family of tyrosine kinase regulatory genes is an ideal target for studies of disease progression in tyrosine kinase driven malignancies.

scholarly journals The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes.

1993 ◽  
Vol 13 (12) ◽  
pp. 7408-7417 ◽  
Author(s):  
L B Vogel ◽  
D J Fujita
Keyword(s):  
Tyrosine Kinases ◽  
Rous Sarcoma Virus ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Rous Sarcoma ◽  
Cell Extracts ◽  
Pi3k Activity ◽  
Sarcoma Virus ◽  
Chicken Embryo Fibroblasts

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.

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