PAK1, PAK1Δ15, and PAK2: similarities, differences and mutual interactions

Abstract

P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1Δ15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1Δ15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition (IPA-3, FRAX597) or depletion (siRNA) on cell-surface adhesion were monitored by real-time microimpedance measurement. Both PAK1Δ15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using coimmunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterocomplexes were also detected. Interaction of PAK1Δ15 or PAK2 with PAK1-full was associated with extensive PAK1Δ15/PAK2 cleavage. The impedance measurements indicate, that PAK2 depletion slows down cell attachment to a surface, and that PAK1-full is involved in cell spreading. Altogether, our data suggest a complex interplay among different PAK group I members, which have non-redundant functions.