Selective targeting of cortactin tandem repeat acetylation by human lysine deacetylases

Lysine acetylation within the tandem repeat region of cortactin (CTTN) regulates its actin‐binding function and has been linked to cancer cell migration and neuronal development. While several lysine deacetylases (KDACs) have been implicated in modulating CTTN acetylation in cells, their site specificity and direct enzymatic roles remain poorly defined. Here, we use genetic code expansion to generate seven site‐specifically acetylated CTTN variants and assess their deacetylation by human KDACs in a fully reconstituted in vitro system. Our results identify HDAC6 as the primary CTTN deacetylase, acting via its second catalytic domain (DD2), and demonstrate that SIRT1 and SIRT2 also directly deacetylate CTTN at overlapping sites in an NAD + ‐dependent manner. In contrast, other zinc‐dependent HDACs, including HDAC8, displayed negligible or very weak activity on full‐length CTTN. These findings provide new mechanistic insight into KDAC substrate preferences and highlight the value of biochemical reconstitution for dissecting complex acetylation networks.