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Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60c-src activity

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Abstract

pp60c-srckinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60c-src but failed to increase hormone independent (basal) pp60c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60c-src was reduced 54±16% compared to controls. Hormone-independent pp60c-src kinase activity was unchanged by expression of the PTPase. pp60c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition,in vitro dephosphorylation by CD45 increased pp60c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60c-src was not. The lack of activation of pp60c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.

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Abbreviations

PTPase:

phosphotyrosine phosphatase

PDGF:

platelet-derived growth factor

PMSF:

phenylmethylsulfonyl fluoride

LCA, CD45:

leukocyte common antigen

PBS:

phosphate buffered saline

SDS:

sodium dodecyl sulfate

PAGE:

polyacrylamide gel electrophoresis

DTT:

dithiothreitol

Na3VO4 :

sodium orthovanadate

PV:

pervanadate

β-ME:

β-mercaptoethanol

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., 14642, Rochester, New York, USA

    Barbara A. Way & Robert A. Mooney

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  1. Barbara A. Way
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  2. Robert A. Mooney
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Way, B.A., Mooney, R.A. Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60c-src activity. Mol Cell Biochem 139, 167–175 (1994). https://doi.org/10.1007/BF01081740

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  • DOI: https://doi.org/10.1007/BF01081740

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