Electron microphotographs and Western blots of control and caveolin-1 and/or caveolin-2 expressing LNCaP and MDCK cells. Two days after infection with adenoviruses encoding β-galactosidase (Adβ-gal; A), myc-tagged caveolin-1 (Adcav-1; B), or both myc-tagged caveolin-1 and -2 (C), cells were processed for electron microscopy or lysed with lysis buffer and subjected to Western blot analysis with caveolin antibodies (F). (D) Caveolae of MDCK cells for comparison. (E) Higher magnification of uncoated vesicles induced by Adcav-1 and caveolae transduced with Adcav-1 and -2 in LNCaP and MDCK cells for comparison. (F) Expression of caveolin-1 and -2 proteins determined by Western blotting in lysates of LNCaP cells infected with multiplicity of infection of viruses identical to that in cells subjected to electron microscopy as compared with MDCK cells, which express ample amounts of caveolin-2 (29); however, the mAb (Transduction Laboratories) that we used does not recognize canine caveolin-2 (lane 6). Lysates were blotted with caveolin-1 pAb (cav-1, Top), caveolin-2 mAb (cav-2, Middle), and β-actin mAb (actin, Bottom). Arrows denote caveolae (C, D, and E Center and Right). Arrowheads denote uncoated uncoated vesicles (B, C, and E Left). (Scale bars, 100 nm.)

Phospho-serine-specific caveolin-2 antibodies reveal that caveolin-2 is phosphorylated in vivo at both serines 23 and 36 with the possible involvement of CK2. (A) The immunoblots of cell lysates from LNCaP cells expressing wild-type and mutant forms of caveolin-2 are shown. (B) Cells expressing caveolin-2 were lysed, immunoprecipitated with caveolin-2 mAb, control (lane 1), subjected to CK2 assay (lane 2), or treated with CIP at 37C (lane 3), and Western blotted with P-Ser 23 (Top), stripped, and reblotted with P-Ser 36 (Middle) pAbs, followed by reblotting with a monoclonal caveolin-2 antibody (Total cav-2; Bottom). (C) Cells expressing caveolin-2 were treated with 30 μM DRB at indicated times, and lysates containing equal amount of caveolin-2 were resolved and Western blotted as described in B.

Phosphorylation of caveolin-2 in vivo and in vitro. (A) The putative CK 2 phosphorylation sites is conserved. The asterisks denote the potential phosphorylation sites and lines represent consensus sites for CK2. (B) The incorporation of ³²P into immunoprecipitated wild-type caveolin-2 and various mutants is shown. LNCaP cells were transiently transfected with the cDNAs (Upper), followed by immunoblotting for caveolin-2 (Lower). (C) The distribution of radio-active peptides by reverse-phase HPLC is shown. The arrows depict fractions in which tryptic peptides containing serines 23 and 36 were detected by mass spectrometry. (D) ³²P labeling of caveolin-2 and its mutated forms expressed as GST-fusion proteins were subjected to in vitro phosphorylation by CK2 (Top) and stained with SBSS (Middle). (Botom) Quantitative analysis of labeling data, normalized to ³²P incorporation into wild-type caveolin-2.

Subcellular targeting and biophysical properties of S23/36/135A remain indistinguishable from wild-type caveolin-2. (A) Parental (Left) or stably expressing caveolin-1 clone 5 of LNCaP cells (Right) were fixed and labeled with caveolin-2 mAb. Arrowheads point to perinuclear caveolin-2 in LNCaP cells expressing caveolin-2 only. Arrows denote examples of plasma membrane caveolin-2 in LNCaP cells stably expressing caveolin-1. (Scale bar, 5 μm.) (B) LNCaP cells expressing either wild-type or S23/36/135A caveolin-2 were lysed in SDS/PAGE loading buffer and boiled (+) or left at room temperature for 30 min (–), and proteins were resolved on a 3–17% gradient SDS/PAGE and subsequently Western blotted with caveolin-2 and 1 Abs. Arrowheads point to oligomers, bands migrating above 200 kDa, and monomers, bands below 33 kDa, of caveolins. (C) Cells expressing wild-type or S23/36/135A caveolin-2 were lysed with MBS buffer (pH 6.5) containing 1% Triton X-100 and centrifuged, and pellets were resuspended with equal volumes of SDS/PAGE loading buffer and Triton X-100 soluble (S) and insoluble (I) proteins resolved on a SDS/13% PAGE, and subsequently Western blotted with caveolin-2 and -1 antibodies.