Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin α6β4

The laminin-binding integrin alpha 6 beta 4 plays key roles in both normal epithelial and endothelial cells and during tumor cell progression, metastasis, and angiogenesis. Previous cysteine mutagenesis studies have suggested that palmitoylation of alpha 6 beta 4 protein supports a few integrin-dependent functions and molecular associations. Here we took another approach and obtained strikingly different results. We used overexpression and RNAi knockdown in multiple cell types to identify protein acyl transferase DHHC3 as the enzyme responsible for integrin beta 4 and alpha 6 palmitoylation. Ablation of DHHC3 markedly diminished integrin-dependent cellular cable formation on Matrigel, integrin signaling through Src, and beta 4 phosphorylation on key diagnostic amino acids (S1356 and 1424). However, unexpectedly, and in sharp contrast to prior alpha 6 beta 4 mutagenesis results, knockdown of DHHC3 accelerated the degradation of alpha 6 beta 4, likely due to an increase in endosomal exposure to cathepsin D. When proteolytic degradation was inhibited (by Pepstatin A), rescued alpha 6 beta 4 accumulated intracellularly, but was unable to reach the cell surface. DHHC3 ablation effects were strongly selective for alpha 6 beta 4. Cell-surface levels of similar to 10 other proteins (including alpha 3 beta 1) were not diminished, and the appearance of hundreds of other palmitoylated proteins was not altered. Results obtained here demonstrate a new substrate for the DHHC3 enzyme and provide novel opportunities for modulating alpha 6 beta 4 expression, distribution, and function.