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.

Group I p21-activated kinases in leukemia cell adhesion to fibronectin

“…The efficiency of IPA-3 and FRAX597 in inhibiting PAK kinase activity was compared using the extent of dephosphorylation at Ser144/141 after 1h cell treatment (examples are given in Supplementary Figure S1). In agreement with our previous results in adherent cells (29), one of the phospho-PAK1 bands, denoted as pPAK1low, was reduced at lower IPA-3 doses then the others. The intensity of pPAK1-low in the cell lines decreased by at least 90% after 1h treatment with 20 µM IPA-3, whereas the other bands were much less reduced (Fig.…”

“…Similar results were obtained in repeated experiments (at least 3 for each cell line and condition). In agreement with the adherent-like phenotype, HEL cell response to IPA-3 or FRAX597 was similar to that of adherent HEK-293T cells (29). In the first setting ( Fig.…”

“…The extent of phosphorylation at pSer144/141 depends on PAK1/PAK2 kinase activity. As we have reported previously (29), the exogenous PAK1 forms are predominantly unphosphorylated. Figure 1B shows PAK expression in AML-derived cell lines in comparison with primary mononuclear cells from AML patients.…”

“…7B, top right). As we have reported earlier, the fast drop upon IPA-3 treatment in adherent and semi-adherent cells was associated with cell contraction (29,34). Microscopic examination of OCI-AML3 cells treated with FRAX597 confirmed that the increase in ECIS signal was due to an increase in the cell-surface contact area ( Fig.…”

“…PAK expression was analyzed using previously characterized antibodies against PAK1, PAK2, or the autophosphorylation site pSer144/141 on PAK1/PAK2, respectively (29). The band pattern obtained from leukemia cells was similar to that from adherent cells: PAK2 antibody recognizes a dominant band at 60 kDa, whereas PAK1 is detected in several bands in the range from 64 to 67 kDa (Fig.…”