Identification of p90, a Prominent Tyrosine-phosphorylated Protein in Fibroblast Growth Factor-stimulated Cells, as 80K-H

doi:10.1074/jbc.271.10.5832

Identification of p90, a Prominent Tyrosine-phosphorylated Protein in Fibroblast Growth Factor-stimulated Cells, as 80K-H (*)

From the Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore

^¶ To whom correspondence should be addressed. Tel.: 65-772-3794; Fax: 65-779-1117; mcbgg{at}leonis.nus.sg.

Abstract

Tyrosine phosphorylation of cellular proteins occurs rapidly upon treatment of fibroblasts with acidic or basic fibroblast growth factors (aFGF, bFGF), suggesting a role for protein phosphorylation in the FGF signaling pathway. Stimulation of Swiss 3T3 cells and MRC-5 fibroblasts with bFGF results in the tyrosine phosphorylation of several proteins, of which the most prominent has been designated as p90. The phosphorylation of p90 is observed within 30 s of treating the cells with FGF but not with other growth factors. Microsequencing of p90 resolved on two-dimensional polyacrylamide gel electrophoresis indicated an N-terminal amino acid sequence which corresponded to a protein previously named as 80K-H. Polyclonal antibodies raised against the predicted C terminus of 80K-H recognized p90 on all Western blots. p90 was found to bind specifically to GRB-2-glutathione S-transferase fusion protein and to be immunoreactive with 80K-H antibody. In addition, anti-phosphotyrosine antibodies immunoprecipitated 80K-H from cell lysates of FGF-stimulated but not from control fibroblasts. The biological function of 80K-H is yet unknown. However, from this study and a previous observation of the obligatory dependence of p90 phosphorylation on FGF receptor occupation, it appears that 80K-H is involved in FGF signaling.

Footnotes

↵^§ These authors have contributed equally to this work.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

FGF

fibroblast growth factor

PAGE

polyacrylamide gel electrophoresis

CSF-1

colony stimulating factor-1

ECL

enhanced chemiluminescence

EGF

epidermal growth factor

bFGF

basic fibroblast growth factor

aFGF

acidic fibroblast growth factor

FGFR-1

fibroblast growth factor receptor type 1

FGFR-2

fibroblast growth factor receptor type 2

FGFR-3

fibroblast growth factor receptor type 3

FGFR-4

fibroblast growth factor receptor type 4

GST

glutathione S-transferase

IL-1

interleukin 1

IL-6

interleukin 6

IRS

insulin receptor substrate

MARCKS

myristoylated alanine-rich C kinase substrate

PBS

phosphate-buffered saline

PDGF

platelet-derived growth factor

PTP1D

protein tyrosine phosphatase type 1D (Syp)

PVDF

polyvinylidene difluoride

SH2

Src homology 2

SH3

Src homology 3

PMSF

phenylmethylsulfonyl fluoride.
- Received October 11, 1995.