Tyrosine Phosphorylation of Caveolin-2 at Residue 27: Differences in the Spatial and Temporal Behavior of Phospho-Cav-2 (pY19 and pY27)

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• 作者：Xiao Bo Wang ; Hyangkyu Lee ; Franco Capozza ; Shana Marmon ; Federica Sotgia ; James W. Brooks ; Roberto Campos-Gonzalez ; and Michael P. Lisanti
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：November 2, 2004
• 年：2004
• 卷：43
• 期：43
• 页码：13694 - 13706
• 全文大小：2478K
• 年卷期：v.43,no.43(November 2, 2004)
• ISSN：1520-4995

文摘

Caveolin-2 is an accessory molecule and the binding partner of caveolin-1. Previously, weshowed that c-Src expression leads to the tyrosine phosphorylation of Cav-2 at position 19. To furtherinvestigate the tyrosine phosphorylation of Cav-2, we have now generated a novel phospho-specific antibodydirected against phospho-Cav-2 (pY27). Here, we show that Cav-2 is phosphorylated at both tyrosines 19and 27. We reconstituted this phosphorylation event by recombinantly coexpressing c-Src and Cav-2. Wegenerated a series of Cav-2 constructs harboring the mutation of each tyrosine to alanine, singly or incombination, i.e., Cav-2 Y19A, Y27A, and Y19A/Y27A. Recombinant expression of these mutants inCos-7 cells demonstrated that neither tyrosine is the unique phosphorylation site, and that double mutationof tyrosines 19 and 27 to alanine abrogates Cav-2 tyrosine phosphorylation. Immunofluorescence analysisof NIH 3T3 cells revealed that the two tyrosine-phosphorylated forms of Cav-2 exhibited some distinctproperties. Phospho-Cav-2 (pY19) is concentrated at cell edges and at cell-cell contacts, whereas phospho-Cav-2 (pY27) is distributed in a dotlike pattern throughout the cell surface and cytoplasm. Further functionalanalysis revealed that tyrosine phosphorylation of Cav-2 has no effect on its targeting to lipid rafts, butclearly disrupts the hetero-oligomerization of Cav-2 with Cav-1. In an attempt to identify upstreammediators, we investigated Cav-2 tyrosine phosphorylation in an endogenous setting. We found that inA431 cells, EGF stimulation is sufficient to induce Cav-2 phosphorylation at tyrosines 19 and 27. However,the behavior of the two phosphorylated forms of Cav-2 diverges upon EGF stimulation. First, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) display different localization patterns. In addition, the temporalresponse to EGF stimulation appears to be different. Cav-2 is phosphorylated at tyrosine 19 in a rapidand transient fashion, whereas phosphorylation at tyrosine 27 is sustained over time. Three SH2 domain-containing proteins, c-Src, Nck, and Ras-GAP, were found to associate with Cav-2 in a phosphorylation-dependent manner. However, phosphorylation at tyrosine 27 appears to be more critical than phosphorylationat tyrosine 19 for this binding to occur. Taken together, these results suggest that, in addition to thecommon characteristics that these two sites appear to share, phospho-Cav-2 (pY19) and phospho-Cav-2(pY27) may each possess a set of unique functional roles.