DHHC2 affects palmitoylation, stability, and functions of tetraspanins CD9 and CD151

Although palmitoylation markedly affects tetraspanin protein biochemistry and functions, relevant palmitoylating enzymes were not known. There are 23 mammalian "DHHC" (Asp-His-His-Cys) proteins, which presumably palmitoylate different sets of protein substrates. Among DHHC proteins tested, DHHC2 best stimulated palmitoylation of tetraspanins CD9 and CD151, whereas inactive DHHC2 (containing DH→AA or C→S mutations within the DHHC motif) failed to promote palmitoylation. Furthermore, DHHC2 associated with CD9 and CD151, but not other cell surface proteins, and DHHC2 knockdown diminished CD9 and CD151 palmitoylation. Knockdown of six other Golgi-resident DHHC proteins (DHHC3, -4, -8, -17, -18, and -21) had no effect on CD9 or CD151. DHHC2 selectively affected tetraspanin palmitoylation, but not the palmitoylations of integrin β4 subunit and bulk proteins visible in [³H]palmitate- labeled whole cell lysates. DHHC2-dependent palmitoylation also had multiple functional effects. First, it promoted physical associations between CD9 and CD151, and between α3 integrin and other proteins. Second, it protected CD151 and CD9 from lysosomal degradation. Third, the presence of DHHC2, but not other DHHC proteins, shifted cells away from a dispersed state and toward increased cell- cell contacts.