Serine Phosphorylation-dependent Association of the Band 4.1-related Protein-tyrosine Phosphatase PTPH1 with 14-3-3β Protein

doi:10.1074/jbc.272.43.27281

Serine Phosphorylation-dependent Association of the Band 4.1-related Protein-tyrosine Phosphatase PTPH1 with 14-3-3β Protein*

From the ^‡Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 and ^‖Howard Hughes Medical Institute,^¶Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Abstract

PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeletal-associated proteins. PTPH1 was found to associate with 14-3-3β using a yeast two-hybrid screen, and its interaction could be reconstituted in vitro using recombinant proteins. Examination of the interaction between 14-3-3β and various deletion mutants of PTPH1 by two-hybrid tests suggested that the integrity of the PTP is important for this binding. Although both PTPH1 and Raf-1 form complexes with 14-3-3β, they appear to do so independently. Binding of 14-3-3β to PTPH1in vitro was abolished by pretreating PTPH1 with potato acid phosphatase and was greatly enhanced by pretreating with Cdc25C-associated protein kinase. Thus the association between PTPH1 and 14-3-3β is phosphorylation-dependent. Two novel motifs RSLS³⁵⁹VE and RVDS⁸⁵³EP in PTPH1 were identified as major 14-3-3β-binding sites, both of which are distinct from the consensus binding motif RSXSXP recently found in Raf-1. Mutation of Ser³⁵⁹ and Ser⁸⁵³ to alanine significantly reduced the association between 14-3-3β and PTPH1. Furthermore, association of PTPH1 and 14-3-3β was detected in several cell lines and was regulated in response to extracellular signals. These results raise the possibility that 14-3-3β may function as an adaptor molecule in the regulation of PTPH1 and may provide a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways.

Footnotes

↵* This work was supported in part by National Cancer Institute Grant CA53840 (to N. K. T.) and a National Institutes of Health Training Grant GM18428 (to P. R. G.).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.
↵§ Glaxo Postdoctoral Fellow.
↵** Associate Investigator of the Howard Hughes Medical Institute.
↵‡ To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: 516-367-8846; Fax: 516-367-6812; E-mail: tonks{at}cshl.org.
↵1 The abbreviations used are: PTP, protein-tyrosine phosphatase; C-TAK1, Cdc25C-associated protein kinase; DMEM, Dulbecco’s modified Eagle’s medium; MBP, maltose-binding protein; PAP, potato acid phosphatase; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; EGF, epidermal growth factor; RCML, reduced, carboxamidomethylated, and maleylated lysozyme.
↵2 H. Piwnica-Worms, unpublished data.
↵3 S.-H. Zhang and N. K. Tonks, unpublished data.
- Received April 11, 1997.
- Revision received August 12, 1997.