Biochemical Characterization of a Human Band 4.1-related Protein-tyrosine Phosphatase, PTPH1

doi:10.1074/jbc.270.34.20067

Biochemical Characterization of a Human Band 4.1-related Protein-tyrosine Phosphatase, PTPH1 (*)

From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208

^§§Pew Scholar in the Biomedical Sciences. To whom correspondence should be addressed:
Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724-2208.
Tel.: 516-367-8846; Fax: 516-367-6812; Tonks{at}cshl.org

↵^¶ Supported by the Council for Tobacco Research, U.S.A. Inc. Present address: Dept. of Biology, Howard University, Washington, D. C. 20059.
↵** Present address: Gene Therapy Center, 944 Wilson Hall CB#7352, University of North Carolina, Chapel Hill, NC 27599.

Abstract

PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeleton-associated proteins. Here, we report the purification and biochemical characterization of this enzyme from baculovirus-infected insect cells. The purified protein exhibited an apparent M_r of 120,000 on SDS gels. The native enzyme dephosphorylated both myelin basic protein (MBP) and reduced, carboxamidomethylated, and maleylated lysozyme (RCML) but was over 5-fold more active on MBP. The K values for the two substrates were similar (1.45 μM for MBP and 1.6 μM for RCML). Phosphorylation of PTPH1 by protein kinase C in vitro resulted in a decrease in K but had no effect on V_max. Removal of the NH₂-terminal band 4.1 homology domain of PTPH1 by limited trypsin cleavage stimulated dephosphorylation of RCML but inhibited its activity toward MBP. The dephosphorylation of RCML by full-length PTPH1 was enhanced up to 6-fold by unphosphorylated MBP and increasing ionic strength up to 0.2 M NaCl, whereas trypsinized preparations of PTPH1 containing the isolated catalytic domain were unaffected. These results suggest that in addition to a potential role in controlling subcellular localization, the NH₂-terminal band 4.1 homology domain of PTPH1 may exert a direct effect on catalytic function.

Footnotes

↵^§ Cold Spring Harbor Association Fellow.
↵* This work was supported by Grant CA53840 from the National Cancer Institute. 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:

PTP

protein-tyrosine phosphatase

DTT

dithiothreitol

FFQ

Fast Flow Q

MAP

mitogen-activated protein

MBP

myelin basic protein

RCML

reduced, carboxamidomethylated, and maleylated lysozyme

PAGE

polyacrylamide gel electrophoresis.
- Received November 1, 1994.
- Revision received June 5, 1995.