The type I PAKs are functionally and structurally well-studied, and are directly activated by BEZ235 interaction with Rho-family small GTPases to function in growth factor signaling and regulation of morphogenic processes. In contrast, the type II PAKs bind the Rho-family small GTPases CDC42, RAC1 and RhoV, but are not directly activated by this interaction. Instead, 
alternate mechanisms of activation and regulation have recently been discovered. The type II PAKs are important for signaling LY294002 cascades that regulate cell survival, neurite outgrowth and formation of filipodia. PAK6 is expressed in prostate, testis, thyroid, placenta and neural tissues and is found in both cytoplasmic and nuclear fractions of prostate cells. Androgen receptor is reported to be a downstream target of PAK6, and PAK6 can regulate gene transcription by androgen receptor via a GTPase-independent mechanism possibly related to control of its degradation by the MDM2 E3 ubiquitin ligase. Global deletion of Pak6 in mice results in increased weight and decreased aggression, possibly explained by its role in androgen receptor signaling. In addition, mice with combined deletion of Pak5 and Pak6 show deficits in locomotion, learning and memory not associated with single deletions of either gene, suggesting functional redundancy between the two PAKs. While neuronal substrates specific to PAK6 have not been identified, PACSIN1, an FBAR protein involved in synaptic vesicle recycling, is phosphorylated redundantly by PAK4, PAK5 and PAK6 in vivo PAK6 is overexpressed in prostate cancer, and its targeted inhibition could potentially decrease growth of prostate tumors or sensitize prostate cancer cells to radiotherapy. PAK6 has also been found to acquire somatic mutations in other solid tumors, including mutation of residue Pro52 to leucine in two independent melanomas. Consequently there is increasing interest in obtaining an improved understanding the various roles of PAK6 in the cell, its substrates and autoregulation, its importance in disease and its potential targeted inhibition. Regulation of type II PAKs was poorly understood until recently. Unlike many protein kinases where phosphorylation at conserved sites within the so-called ��activation loop�� is a critical step towards full activity, the type II PAKs are constitutively phosphorylated in the cell and not directly regulated by interaction with small GTPases, which are instead important for type II PAK relocalization. We, and others, identified an autoinhibitory sequence within the N-terminal region of PAK4 and showed by structural and biochemical analysis that this region contains a pseudosubstrate sequence centered around residue P52. Based on this work, we hypothesized that this highly conserved N-terminal region could autoinhibit each of the type II PAKs. ATP-competitive small molecule inhibitors of the type II PAKs could be useful as cancer therapeutics. The small molecule PF-3758309 was designed as a PAK4-specific inhibitor, but displays in vitro activity against each of the type II PAKs and also PAK1. Though effective in mouse models of cancer, it failed in human clinical trials. Sunitinib is a potent ATPcompetitive multi-kinase inhibitor that is indicated for treatment of renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumors, advanced pancreatic neuroendocrine tumors and other tumor types. A crystal structure is available for PAK4 with PF-3758309 but none are available for a PAK family member in complex with sunitinib. In the current study we ask whether downstream substrate specificity is conserved among the type II PAKs, whether a cancerassociated mutation that occurs in the type II PAK autoinhibitory region can activate PAK6, and whether co-crystallography might aid drug discovery for type II PAKs. By peptide array profiling we show that PAK6 has a similar substrate specificity to that previously observed for PAK4 and PAK5, implying that PAK6 may have additional substrates that overlap with other type II PAKs. We show that PAK6 kinase activity is regulated by its Nterminal pseudosubstrate in vitro and that a melanoma-associated mutation, P52L, in the pseudosubstrate sequence displays reduced inhibition. We go on to determine crystal structures of PAK6 kinase domain in complex with two ATP-competitive small molecule inhibitors, PF-3758309 and sunitinib. This study therefore provides molecular level details that may aid in the development of isotype specific inhibitors for the type II PAKs.