Substrate and Inhibitor Specificity of the Type II p21-Activated Kinase, PAK6

Abstract

The p21-activated kinases (PAKs) are important effectors of Rho-family small GTPases. The PAK family consists of two groups, type I and type II, which have different modes of regulation and signaling. PAK6, a type II PAK, influences behavior and locomotor function in mice and has an ascribed role in androgen receptor signaling. Here we show that PAK6 has a peptide substrate specificity very similar to the other type II PAKs, PAK4 and PAK5 (PAK7). We find that PAK6 catalytic activity is inhibited by a peptide corresponding to its N-terminal pseudosubstrate. Introduction of a melanoma-associated mutation, P52L, into this peptide reduces pseudosubstrate autoinhibition of PAK6, and increases phosphorylation of its substrate PACSIN1 (Syndapin I) in cells. Finally we determine two co-crystal structures of PAK6 catalytic domain in complex with ATP-competitive inhibitors. We determined the 1.4 Å co-crystal structure of PAK6 with the type II PAK inhibitor PF-3758309, and the 1.95 Å co-crystal structure of PAK6 with sunitinib. These findings provide new insights into the structure-function relationships of PAK6 and may facilitate development of PAK6 targeted therapies.

PAK6 targets to cell-cell adhesions via its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape

“…2A,B) to test whether PAK6 catalytic activity could affect its ability to localize to cell-cell adhesions. We found that kinase-dead PAK6 K436M (Schrantz et al, 2004) and hyperactive PAK6 P52L (Gao et al, 2013) both localize to cell-cell adhesions, as identified either by F-actin staining (Fig. 2C) or β-catenin staining (Fig.…”

“…PAK6 requires a functional CRIB motif to target to cell-cell adhesions Few protein domains or motifs are ascribed to the PAK6 N-terminus (residues 1-382) with the exceptions of a polybasic (PB) region (residues 3-9), a well-conserved CRIB motif (residues 12-25), and a pseudosubstrate region that inhibits catalytic activity (residues 48-56) (Ha et al, 2012;Gao et al, 2013;Wang et al, 2013). Having found PAK6 cell-cell adhesion targeting is not kinase activity dependent, we focused our attention on the polybasic region and CRIB motif as potential requirements for targeting.…”

Signaling, Regulation, and Specificity of the Type II p21-activated Kinases

“…Further, PAK6 is autoregulated by the pseudosubstrate region and can be activated in cells by mutation of Pro 52 (56). Another study also used x-ray crystallography to observe an identical pseudosubstrate conformation for the interaction of a longer peptide fragment with the PAK4 kinase domain (42).…”

“…In contrast, the type II PAKs do not follow this paradigm and are observed to be constitutively phosphorylated in cells (35,47). Type II PAKs are consistently activation loop-phosphorylated when produced in Escherichia coli for structural studies (42,46,56,62), and although PAK4 activation loop phosphorylation has been used as a marker for activation in cell-based assays and as a histological marker (19,(63)(64)(65)(66), the validity of this readout has been recently questioned (47). Therefore, the role of modulating phosphorylation in the activation loop of the type II PAKs as a regulatory mechanism is not completely clear.…”

“…Interestingly, the mutation (T538N) maps to the catalytic cleft, suggesting release of pseudosub-strate inhibition as a potential mechanism (87). A recurrent PAK6 mutation at the central Pro residue in the pseudosubstrate region (P52L) found in melanomas (90,91) resulted in increased catalytic activity (56). Further studies will be required to investigate whether, or how, other cancer-associated mutations impact catalytic activity or other PAK functions.…”