Identification and Cloning of Xp95, a Putative Signal Transduction Protein in Xenopus Oocytes

doi:10.1074/jbc.274.9.5522

Identification and Cloning of Xp95, a Putative Signal Transduction Protein in Xenopus Oocytes*

From the^‡Departments of Clinical Investigation, ^§Molecular Genetics, and ^¶Experimental Radiation Oncology, the University of Texas, M. D. Anderson Cancer Center, Houston and the ^‖Department of Pharmacology, the University of Texas Medical School, Houston, Texas 77030

Abstract

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and AspergillusPalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA intoXenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.

Footnotes

↵* This work was supported by National Cancer Institute Grant R-29 GM48457–02, National Science Foundation Grant MCB-9405699 (to J. K.), and National Cancer Institute Training Grant CA09299 (to H. E.-H.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF115497.
↵** To whom correspondence should be addressed: Dept. of Clinical Investigation, Box 019, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-2961; Fax: 713-792-3754; E-mail:jkuang{at}notes.mdacc.uth.tmc.edu.
Abbreviations:

MAP

mitogen-activated protein

DTT

dithiothreitol

EB

Xenopusegg extraction buffer

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

TBST

Tris-based saline/Tween 20

PCR

polymerase chain reaction

ATPγS

adenosine 5′-O-(thiotriphosphate)

d-Src

deregulated Src

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
- Received September 28, 1998.
- Revision received November 9, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.