Ligand Activation of ELK Receptor Tyrosine Kinase Promotes Its Association with Grb10 and Grb2 in Vascular Endothelial Cells

doi:10.1074/jbc.271.38.23588

Ligand Activation of ELK Receptor Tyrosine Kinase Promotes Its Association with Grb10 and Grb2 in Vascular Endothelial Cells*

From the Departments of Pharmacology and
^§ Cell Biology and the
^¶ Division of Nephrology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and
^‡ Immunex Corporation, Seattle, Washington 98101

^∥ To whom correspondence should be addressed:
Div. of Nephrology, MCN S3223, Vanderbilt University School of Medicine, 1116 Garland St., Nashville, TN 37232
. Tel.: 615-343-9867; Fax: 615-343-7156; E-mail: daniel{at}mbio.mc.vanderbilt.edu

Abstract

ELK is a member of the Eph-related tyrosine kinase family that includes receptors signaling axonal guidance, neuronal bundling, and angiogenesis. We recently identified ELK expression in human renal microvascular endothelial cells and sought to identify intracellular proteins through which it signals responses. The cytoplasmic domain of ELK was used as “bait” in a yeast two-hybrid screen to identify interactive proteins expressed from a randomly primed embryonic murine library (E9.5-10.5). Among interactive products of 76 cDNAs characterized, 10 nonidentical, overlapping clones encoded the SH2 domain of the recently reported Grb10 adapter protein, and an additional 3 encoded Grb2. A self-phosphorylated recombinant, baculovirus-expressed GST-ELK_cy fusion protein bound Grb10 and Grb2 from human renal microvascular endothelial cell extracts, while the unphosphorylated fusion form did not. Site-directed mutation identified Tyr-929 as a putative phosphorylation site required for Grb10, but not Grb2, interaction in yeast and recombinant protein assays. The ELK ligand, LERK-2/Fc, stimulated tyrosine phosphorylation of ELK, and recruitment of Grb10 and Grb2 to endothelial ELK receptors recovered by wheat germ agglutinin lectin and immunoprecipitation. These findings define ligand-activated interaction between ELK and the SH2 domains of Grb2 and the newly identified Grb10 protein that shares homology with a Caenorhabditis elegans gene product implicated in neural cell migration.

Footnotes

↵* This work was supported by Grants DK38517 and DK47078 from the United States Public Health Service (to T. O. D.) and the Renal Care Group. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ E. Stein, H. O. Schoecklmann, D. P. Cerretti, and T. O. Daniel, submitted for publication.
↵² E. Stein and T. O. Daniel, unpublished results.
↵³ The abbreviations used are:

ELK_cy

cytoplasmic domain of HuELKI (amino acids 566-984)

HREMC

human renal microvascular endothelial cells

PCR

polymerase chain reaction

GST

glutathione S-transferase

ELK_cyΔCterm

COOH-terminal domain deletion of ELK_cy (retaining amino acids 566-883)

ELK_cyΔJM

juxtamembrane deletion of ELK_cy (retaining amino acids 618-984)

ELK_cyK652R

site-directed mutant substituting lysine at position 652 with arginine

ELK_cyY929F

site-directed mutant substituting tyrosine at position 929 with phenylalanine

LERK-2/Fc

a fusion protein including the extracellular domain of LERK-2 and IgG1 Fc domain

ORF/Fc

a fusion protein including an irrelevant open reading frame and the IgG1 Fc domain

EIP

ELK-interactive protein

PMSF

phenylmethylsulfonyl fluoride.
↵⁴ E. Stein and T. O. Daniel, manuscript in preparation.
↵⁵ J. Manser, unpublished results.
- Received February 23, 1996.
- Revision received June 25, 1996.