Elsevier

Experimental Cell Research

Volume 288, Issue 1, 1 August 2003, Pages 110-118
Experimental Cell Research

Regular article
Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk

https://doi.org/10.1016/S0014-4827(03)00206-4Get rights and content

Abstract

We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.

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)

Cited by (37)

  • Phosphorylation of the activation loop tyrosine 823 in c-Kit is crucial for cell survival and proliferation

    2013, Journal of Biological Chemistry
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    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.

  • Adaptor protein Lnk binds to and inhibits normal and leukemic FLT3

    2012, Blood
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    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

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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.

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