We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

RAC1 is a small, Ras-related GTPase that was recently reported to harbor a recurrent UV-induced signature mutation in melanoma, resulting in substitution of P29 to serine (RAC1 P29S ), ranking this the third most frequently occurring gain-of-function mutation in melanoma. Although the Ras family GTPases are mutated in about 30% of all cancers, mutations in the Rho family GTPases have rarely been observed. In this study, we demonstrate that unlike oncogenic Ras proteins, which are primarily activated by mutations that eliminate GTPase activity, the activated melanoma RAC1 P29S protein maintains intrinsic GTP hydrolysis and is spontaneously activated by substantially increased inherent GDP/GTP nucleotide exchange. Determination and comparison of crystal structures for activated RAC1 GTPases suggest that RAC1 F28L-a known spontaneously activated RAC1 mutant-and RAC1P29S are self-activated in distinct fashions. Moreover, the mechanism of RAC1 P29S and RAC1 F28L activation differs from the common oncogenic mutations found in Ras-like GTPases that abrogate GTP hydrolysis. The melanoma RAC1 P29Sgain-of-function point mutation therefore represents a previously undescribed class of cancer-related GTPase activity.cancer | cell signaling | x-ray crystallography | cytoskeleton | GEF-independent GTPase exchange R AC1, the Ras-related small GTPase belonging to the Rho family, functions as a binary molecular switch cycling between an inactive GDP-bound "OFF" state and an active GTP-bound "ON" state (1). Its activity is responsible for the regulation of diverse cellular behaviors including NADPH oxidase activation, formation of cortical actin-containing membrane ruffles and lamellipodia, and induction of gene expression programs (2). Accordingly, these functions are tightly controlled through RAC1 lipidation, subcellular localization, protein expression levels, and Rho GDP-dissociation inhibitor (Rho GDI) interactions. In addition, as a GTPase, RAC1 is turned ON by guanine nucleotide exchange factors (GEFs) and is turned OFF by GTPase activating proteins (GAPs) that facilitate GDP/GTP nucleotide exchange and GTP hydrolysis, respectively. Once this regulation is compromised, RAC1 activity is implicated in various steps of oncogenesis including initiation, progression, invasion, and metastasis (3, 4).In contrast to Ras, RAC1 has rarely been identified as significantly mutated in cancer. Instead, overexpression of RAC1 has been reported in colorectal, pancreatic, breast, and testicular cancers and in various leukemias (5-7). Additionally, a self-activating splice variant of RAC1, RAC1b, was shown to be overexpressed in breast cancer and lung cancer and is thought to mediate the epithelial-mesenchymal transition in lung epithelial cells (8-10). Furthermore, aberrant activation of upstream regulators of RAC1, particularly in the DBL family of GEFs specific for RAC1 (e.g., TIAM1, PREX1, and ECT2), have been implicated in various cancers (11). Although increased GDP→GTP nucleotide exchange in RAC1 (dependent or independent of GEFs) is...

SummaryEukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the “DFG+1” residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.

The type II p21-activated kinases (PAKs) are key effectors of RHOfamily GTPases involved in cell motility, survival, and proliferation. Using a structure-guided approach, we discovered that type II PAKs are regulated by an N-terminal autoinhibitory pseudosubstrate motif centered on a critical proline residue, and that this regulation occurs independently of activation loop phosphorylation. We determined six X-ray crystal structures of either fulllength PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop. We show that full-length PAK4 is constitutively autoinhibited, but mutation of the pseudosubstrate releases this inhibition and causes increased phosphorylation of the apoptotic regulation protein Bcl-2/Bcl-X L antagonist causing cell death and cellular morphological changes. We also find that PAK6 is regulated by the pseudosubstrate region, indicating a common type II PAK autoregulatory mechanism. Finally, we find Src SH3, but not β-PIX SH3, can activate PAK4. We provide a unique understanding for type II PAK regulation.autoregulation | protein kinase | RHO GTPase effector | signaling T he RHO-family small GTPases RAC1 and CDC42 control many cellular functions, including cytoskeletal organization, morphological changes, cell motility, and cell-cycle progression (1). These enzymes are regulated by cycling between GDPbound and GTP-bound states, whereby the GTP-bound state binds to and activates multiple effector molecules, triggering distinct downstream events. Pathological mutations in these proteins can alter cellular outcomes, and recurrent activating mutations suffer high mutational burdens in cancer (2). There is consequently significant interest in understanding the intrinsic details of these molecules and their complex signaling pathways.An important group of proteins that directly interact with RAC1 and CDC42 are the p21-activated kinases (PAKs) (3). These Ste20 family serine-threonine kinases are regulators of the actin cytoskeleton, cell survival, cell adhesion, cytokine signaling, and transcription (3, 4). There are two subgroups of PAK kinase, denoted type I (PAK1, PAK2, and PAK3) and type II (PAK4, PAK5, and PAK6). PAK4 is the best-studied type II PAK family member, is widely expressed (5), and is essential for viability in mice (6). Downstream substrates of PAK4 include the RHO GTPase guanine nucleotide exchange factor H1 (7), the cytoskeletal regulator LIM domain kinase 1 (LIMK1) (8), the adhesion receptor integrin β5 (9), the focal adhesion scaffolding protein paxillin (10), and the apoptotic regulation protein Bcl-2/ Bcl-X L antagonist causing cell death (BAD) (11). This array of substrates are thought to mediate effects of PAK4 activation in cells, including loss of focal adhesions (12), cell rounding (12, 13), cytoskeleton changes (13), and protection from apoptosis (11). Together, these observations indicate that PAK4 plays roles both as a regulator of the actin cytoskeleton and as a promoter of ...

Ubiquitin-fold modifier 1 (Ufm1)-specific protease 2 (UfSP2) is a cysteine protease that is responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins, as well as for the generation of mature Ufm1 from its precursor. The 2.6 Å resolution crystal structure of mouse UfSP2 reveals that it is composed of two domains. The C-terminal catalytic domain is similar to UfSP1 with Cys 294 , Asp 418 , His 420 , Tyr 282 , and a regulatory loop participating in catalysis. The novel N-terminal domain shows a unique structure and plays a role in the recognition of its cellular substrate C20orf116 and thus in the recruitment of UfSP2 to the endoplasmic reticulum, where C20orf116 predominantly localizes. Mutagenesis studies were carried out to provide the structural basis for understanding the loss of catalytic activity observed in a recently identified UfSP2 mutation that is associated with an autosomal dominant form of hip dysplasia.