Differential Regulation of Protein-tyrosine Phosphatases by Integrin through Cytoskeletal Reorganization and Tyrosine Phosphorylation in Human Platelets

doi:10.1074/jbc.270.20.11927

Differential Regulation of Protein-tyrosine Phosphatases by Integrin through Cytoskeletal Reorganization and Tyrosine Phosphorylation in Human Platelets (*)

From the (1) First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606, Japan

^§To whom correspondence should be addressed. Tel.: 81-75-751-3151; Fax: 81-75-751-3201.

Abstract

The major platelet integrin αβ₃ (glycoprotein IIb-IIIa) has been implicated in the regulation of tyrosine phosphorylation and dephosphorylation in activated platelets. To investigate the mechanisms of the αβ₃-dependent tyrosine dephosphorylation, normal platelets or thrombasthenic platelets lacking αβ₃ were stimulated with thrombin and fractionated into Triton X-100-soluble or -insoluble subcellular matrices. We then examined the kinetics of the tyrosine-phosphorylated proteins and distribution of protein-tyrosine phosphatases in these fractions and whole cell lysates. First, αβ₃-dependent tyrosine dephosphorylation was recovered mainly in the cytoskeleton with similar kinetics to the whole cell lysate. Second, protein-tyrosine phosphatase (PTP) 1B and its cleaved 42-kDa form were associated with the cytoskeleton in an aggregation-dependent manner, whereas association of PTP1C with the cytoskeleton was regulated differentially both by thrombin stimulation and by αβ₃-mediated aggregation. Several calpain inhibitors did not affect either tyrosine phosphorylation and dephosphorylation or relocation of PTP1B, but they did inhibit cleavage of PTP1B. Cytochalasin D blocked relocation of both PTP1B and PTP1C but not PTP1B cleavage. SH-PTP2 was distributed in the other fractions than the cytoskeleton and showed no relocation on thrombin stimulation. Finally, the cytoskeleton-associated PTP1C became tyrosine-phosphorylated in an αβ₃-mediated aggregation-dependent manner. Thus, integrin αβ₃ was involved differentially in the regulation of PTP1B and PTP1C.

Footnotes

↵* This work was supported by a grant-in-aid for general scientific research from the Ministry of Education, Science and Culture, Japan. This study was presented at the 1994 Annual Meeting of the American Society of Hematology, Nashville, TN and published in abstract form (Ezumi, Y., Takayama, H., Ichinohe, T., Hirai, K., Tomo, K., and Okuma, M. (1994) Blood 84, 321a). 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:

PTK

protein-tyrosine kinase

PTPase

protein-tyrosine phosphatase

SH2

Src homology 2

RGDS

Arg-Gly-Asp-Ser

IgG

immunoglobulin G

PMSF

phenylmethylsulfonyl fluoride

RIPA

radioimmunoprecipitation assay

PAGE

polyacrylamide gel electrophoresis

EST

ethyl (+)-(2S, 3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)]-2-oxiranecarboxylate

MDL

carbamic acid, [1-[[(1-formyl-2-phenylmethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.
↵²Y. Ezumi, H. Takayama, and M. Okuma, unpublished observations.