Identification of Phosphocaveolin-1 as a Novel Protein Tyrosine Phosphatase 1B Substrate † Hyangkyu Lee, ‡,§ Laiping Xie, ‡,§ Yong Luo, | Seung-Yub Lee, § David S. Lawrence, ⊥ Xiao Bo Wang, § Federica Sotgia, § Michael P. Lisanti,* ,§ and Zhong-Yin Zhang* ,§,|,⊥ Departments of Molecular Pharmacology and Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park AVenue, Bronx, New York 10461, and Department of Biochemistry and Molecular Biology, Indiana UniVersity School of Medicine, 635 Barnhill DriVe, Indianapolis, Indiana 46202 ReceiVed August 4, 2005; ReVised Manuscript ReceiVed NoVember 6, 2005 ABSTRACT: Protein tyrosine phosphatase 1B (PTP1B) is implicated in a number of signaling pathways including those mediated by insulin, epidermal growth factor (EGF), and the Src family kinases. The scaffolding protein caveolin-1 is also a participant in these pathways and is specifically phosphorylated on tyrosine 14, when these pathways are activated. Here, we provide evidence that PTP1B can efficiently catalyze the removal of the phosphoryl group from phosphocaveolin-1. Overexpression of PTP1B decreases tyrosine 14 phosphorylation in caveolin-1, while expression of the substrate-trapping mutant PTP1B/ D181A causes the accumulation of phosphocaveolin-1 and prevents its dephosphorylation by endogenous PTPs. We further demonstrate that PTP1B physically associates with caveolin-1. Finally, we show that inhibition of PTP1B activity with a potent and specific small molecule PTP1B inhibitor blocks the PTP1B- catalyzed caveolin-1 dephosphorylation both in vitro and in vivo. Taken together, the results strongly suggest that caveolin-1 is a specific substrate for PTP1B. Identification of caveolin-1 as a PTP1B substrate represents an important new step in further understanding the signaling pathways regulated by PTP1B. Protein tyrosine phosphorylation controls the activity of many signaling proteins and is involved in almost all eukaryotic cell activities. The state of tyrosine phosphory- lation undergoes dynamic and reciprocal regulation by two large enzyme families: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). 1 By adding or removing phosphate groups to or from substrate proteins, protein tyrosine kinases and PTPs play crucial roles in cellular proliferation, differentiation, metabolism, and survival (1, 2). As important as the tyrosine kinases, the PTPs have been implicated in a number of diseases including cancer, diabetes, and inflammation (3). For example, genetic and biochemical evidence indicates that PTP1B is an important negative regulator of insulin- and leptin-mediated pathways (4). In particular, mice lacking PTP1B are hyperresponsive to insulin and resistant to diet-induced obesity (5, 6), implicating PTP1B as a key therapeutic target in the treatment of type 2 diabetes and obesity. However, besides a role in insulin and leptin signaling, PTP1B may also associate with several other physiological and pathological processes, including the suppression of cell transformation by the Neu oncogene (7, 8) and v-Src (9), activation of the Src kinase (10-12), and antagonizing signaling by the EGF receptor (13-16) and the oncoprotein p210 bcr-abl (17). Taken together, these results suggest that PTP1B may be a participant in several signaling pathways. Because PTP1B may be a regulator of multiple signaling pathways and it can both enhance and antagonize a cellular event, the physiological relevance of PTP1B in these processes needs to be established. This is an important prerequisite for the development of PTP1B-based therapeu- tics for type 2 diabetes and obesity. Despite extensive studies, the unique functional contribu- tions made by PTP1B to individual signal pathways have not yet been fully defined. In part, this is due to a lack of full knowledge of the physiological substrates of PTP1B. Our hypothesis is that PTP1B plays diverse roles in signal transduction because it recognizes distinct substrates in different cellular contexts. Consequently, the identification of PTP1B substrates is an essential step toward a complete understanding of the physiological function of this enzyme. The yeast two-hybrid approach in general has proven ineffective for PTP substrate identification because of the requirement for tyrosine phosphorylation (there are no protein tyrosine kinases in yeast). An effective strategy to identify and characterize PTP substrates has been to employ PTP † This work was supported by National Institutes of Health Grant DK68447 and the G. Harold and Leila Y. Mathers Charitable Foundation (to Z.-Y.Z). M.P.L. was supported by grants from National Institutes of Health (NIH) and the American Heart Association (AHA), as well as a Hirschl/Weil-Caulier Career Scientist Award. H.L. and X.B.W. were supported by NIH Graduate Training Program Grant T32- DK07513. * To whom correspondence should be addressed. M.P.L.: phone, (718) 430-8828; fax, (718) 430-8922; e-mail, lisanti@aecom.yu.edu. Z.-Y.Z.: phone, (317) 274-8025; fax, (317) 274-4686; e-mail, zyzhang@iupui.edu. ‡ These two authors contributed equally to this work. § Department of Molecular Pharmacology, Albert Einstein College of Medicine. | Indiana University School of Medicine. ⊥ Department of Biochemistry, Albert Einstein College of Medicine. 1 Abbreviations: PTP, protein tyrosine phosphatase; CSK, C-terminal Src kinase; GST, glutathione S-transferase; pNPP, p-nitrophenyl phosphate; EGF, epidermal growth factor; IR, insulin receptor. 234 Biochemistry 2006, 45, 234-240 10.1021/bi051560j CCC: $33.50 © 2006 American Chemical Society Published on Web 12/13/2005