Regular articleIdentification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk
Introduction
The receptor for stem cell factor (SCF), c-Kit, is a receptor tyrosine kinase and member of subfamily III of the receptor tyrosine kinases. Together with its ligand, SCF, c-Kit is a key controlling receptor for a number of cell types, including hematopoietic stem cells, mast cells, melanocytes, and germ cells. C-Kit is allelic with the W locus in mice and its ligand, SCF, is the product of the Sl locus. Mutation in either of these loci results in several defects in the growth and survival of stem cells of these tissues (for review, see [1], [2]).
Stimulation of the c-Kit receptor with its ligand results in dimerization of receptors, activation of its intrinsic tyrosine kinase activity, and autophosphorylation of c-Kit on tyrosine residues. These phosphorylated tyrosine residues constitute docking sites for Src homology 2 (SH2) domain containing signal transduction molecules, which will thereby be recruited to the receptor and activated, often through tyrosine phosphorylation. A number of tyrosine residues have been described to be autophosphorylated and some of their docking partners identified: Tyr568 (SHP2, Src family tyrosine kinases, CHK), Tyr570 (SHP1), Tyr703 (Grb2), Tyr721 (PI3-kinase), Tyr823, Tyr936 (Grb2, Grb7) [3], [4], [5], [6], [7], [8]. All these sites are true autophosphorylation sites, i.e., the receptor kinase itself is performing these phosphorylations.
Modulation of receptor tyrosine kinases by other kinases has been described in a number of cases. Examples of serine and/or threonine phosphorylation as a negative regulatory event have been described for the hepatocyte growth factor receptor/c-met, for the EGF receptor, and for c-Kit [9], [10], [11], [12]. In addition several tyrosine kinase receptors have been demonstrated to be phosphorylated by nonreceptor tyrosine kinases, such as the EGF receptor [13] and the PDGF β-receptor [14]. In the case of the PDGF β-receptor, it could be demonstrated that phosphorylation of Tyr934 in the second part of the kinase domain led to a modulation of the substrate specificity of the PDGF receptor kinase activity. Thus, a Y934F mutant of the PDGF β-receptor was much more efficient in phosphorylating PLC-γ1 than wild-type receptors. This was not due to any differences in association of PLC-γ1 with the receptors or differences in the overall kinase activity of the receptor.
Since the PDGF β-receptor and c-Kit are quite closely related, the aim of the present investigation was to elucidate whether c-Kit could also become phosphorylated on a tyrosine residue by c-Src and if so, what the functional consequences would be. In this report we show that Tyr900, which corresponds to Tyr934 in the PDGF β-receptor, is phosphorylated by Src family kinases and that the phosphorylated Tyr900 is an association site for Crk-II, most likely via the p85 subunit of PI3-kinase. Ligand stimulation of the wild-type receptor leads to a small but significant increase in Crk-II tyrosine phosphorylation (two- to threefold). In a receptor where Tyr900 had been replaced by a phenylalanine (Y900F) the Crk-II phosphorylation was dramatically reduced. The binding of Crk-II to c-Kit is likely to be transient since no complex could be detected by co-immunoprecipitation. However, using a GST fusion protein of Crk-II an interaction between Crk-II and c-Kit could be demonstrated.
Section snippets
Materials and methods
Modified sequencing grade trypsin was from Promega (Madison, WI). Polyvinylpyrrolidone was from Aldrich (Steinheim, Germany) and soybean trypsin inhibitor–agarose (STI-agarose) from Pierce (Rockford, IL). C-Src kinase, expressed and purified from insect cells, was from Upstate Biotechnology Incorporated (Lake Placid, NY). [35S]Methionine and [35S]cysteine in vivo labeling Promix as well as Redivue [γ-32P]ATP and [32P]orthophosphate (PBS43) were purchased from Amersham (Buckinghamshire, UK). N
Tyr900 is phosphorylated by Src family kinases and not by the intrinsic kinase activity of c-Kit
Porcine aortic endothelial (PAE) cells expressing wild-type c-Kit were incubated with [32P]orthophosphate for 4 h at 37°C and stimulated with 100 ng/ml of recombinant human SCF for 5 min. After lysis and immunoprecipitation of c-Kit, samples were separated by SDS–gel electrophoresis followed by electrotransfer to Hybond C Extra filters. The radiolabeled receptor was digested with modified sequencing grade trypsin, and the resulting tryptic peptides were subjected to immunoprecipitation with a
Discussion
Here we demonstrate that c-Src phosphorylates Tyr900 in the second part of the kinase domain of c-Kit. A receptor mutant in which Tyr900 was replaced with a phenylalanine residue showed a lower degree of association with and phosphorylation of Crk-II than wild-type receptors. In vitro kinase experiments showed that when c-Src was added to immunoprecipitated c-Kit, specific phosphorylation of Tyr900 was seen (Fig. 1). Furthermore, in living cells Tyr900 is phosphorylated in response to SCF
Acknowledgements
Recombinant human SCF was kind gift of AMGEN, Inc. Lars Rönnstrand holds a position as Senior Researcher funded by the Swedish Research Council. This work was partially funded by grants from the Swedish Cancer Society and the Swedish Society for Medical Research.
References (25)
- et al.
Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells
J. Biol. Chem.
(1997) - et al.
Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes
J. Biol. Chem.
(1997) - et al.
Tyrosine residue 719 of the c-kit receptor is essential for binding of the P85 subunit of phosphatidylinositol (PI) 3-kinase and for c-kit-associated PI 3-kinase activity in COS-1 cells
J. Biol. Chem.
(1994) - et al.
Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C
J. Biol. Chem.
(1994) - et al.
Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase
J. Biol. Chem.
(1992) - et al.
Phosphorylation of serine 985 negatively regulates the hepatocyte growth factor receptor kinase
J. Biol. Chem.
(1994) - et al.
Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells
J. Biol. Chem.
(1995) - et al.
c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function
J. Biol. Chem.
(1999) - et al.
The functional role of CrkII in actin cytoskeleton organization and mitogenesis
J. Biol. Chem.
(1999) - et al.
Steel factor induces tyrosine phosphorylation of CRKL and binding of CRKL to a complex containing c-kit, phosphatidylinositol 3-kinase, and p120(CBL)
J. Biol. Chem.
(1997)
Differential interaction of CrkII adaptor protein with platelet-derived growth factor alpha- and beta-receptors is determined by its internal tyrosine phosphorylation
Biochem. Biophys. Res. Commun.
Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis
J. Biol. Chem.
Cited by (37)
Phosphorylation of the activation loop tyrosine 823 in c-Kit is crucial for cell survival and proliferation
2013, Journal of Biological ChemistryCitation Excerpt :Activation of c-Kit by its ligand triggers activation of numerous downstream signal transduction molecules. Some of these molecules are tyrosine kinases by themselves, e.g. Src family kinases such as Lyn and the Fes kinase (21, 22), and have been demonstrated to be able to phosphorylate c-Kit (23). Thus, the phosphorylation status of c-Kit in living cells could be influenced by factors other than the intrinsic kinase activity of c-Kit itself.
Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected
2013, Molecular and Cellular ProteomicsAdaptor protein Lnk binds to and inhibits normal and leukemic FLT3
2012, BloodCitation Excerpt :It is worth noting that Tyr591 of FLT3 and Tyr568 of c-Kit are conserved tyrosines in the JXM domain, the role of which is well defined (supplemental Figure 1). Conversely, the interaction of Lnk with p-Tyr919 is worth investigating further for the following reasons: (1) the binding activity to Lnk is much stronger than the other 2 tyrosines; (2) the conserved residue in murine Flt3 (p-Tyr922) has been shown to be critical for constitutive activation of murine FLT3 mutant41; and (3) the corresponding residues in both c-Kit (p-Tyr900) and the PDGFR-β (p-Tyr934) have been shown to play important roles in maintaining kinase activity.43,44 FLT3 is expressed in almost all human hematopoietic stem cells and HPCs that give rise to lymphoid or myeloid lineages or both.44
Rapid estrogen signalling in mouse primordial germ cells
2010, Experimental Cell ResearchOncogenic signaling from the hematopoietic growth factor receptors c-Kit and Flt3
2009, Cellular Signalling
- 1
Present address: Basic Research laboratory, Division of Basic Sciences, National Cancer Institute, Frederick, MD 21702, USA.
- 2
Present address: Department of Experimental Clinical Chemistry, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden.