Fig. 9. JAM-2 is phosphorylated at serine residue S281 in CHO cells. (A) Phosphoamino acid analysis of JAM-2. CHO cells stably transfected with the S281A mutant of JAM-2 (JAM-2 S281A, left panel) or wild-type JAM-2 (JAM-2 wt, right panel) were metabolically labelled with [³²P]-orthophosphate. Immunoprecipitated JAM-2 was hydrolyzed and the resulting amino acids were subjected to two-dimensional electrophoresis. The broken circles indicate the positions of comigrating cold phosphoamino acids. The inset illustrates the relative positions of free phosphate residues (Pi), phospho-serine (P-Ser), phosphothreonine (P-Thr) and phospho-tyrosine (P-Tyr). JAM-2 is phosphorylated exclusively on serine residues in both cell lines. (B) Two-dimensional phosphotryptic peptide maps of [³²P]-labelled JAM-2 S281A and JAM-2 wt. Immunoprecipitated JAM-2 was subjected to trypsin digestion and the resulting peptides were subjected to electrophoresis and thin layer chromatography as indicated by the arrows. The origins of sample application are indicated by encircled black dots; the position of a marker dye for thin layer chromagtography is indicated by an encircled `M'. The positions of phosphopeptides are indicated by arrowheads. From two phosphopeptides that are identified in wt JAM-2, one is missing in JAM-2 S281A indicating that JAM-2 is phosphorylated at the S281 residue.