Structural and functional characterization of USP47 reveals a hot spot for inhibitor design.

USP47 is widely involved in tumor development, metastasis, and other processes while performing a more regulatory role in inflammatory responses, myocardial infarction, and neuronal development. In this study, we investigate the functional and biochemical properties of USP47, whereby depleting USP47 inhibited cancer cell growth in a p53-dependent manner-a phenomenon that enhances during the simultaneous knockdown of USP7. Full-length USP47 shows higher deubiquitinase activity than the catalytic domain.

Rho family GTPase signaling through type II p21-activated kinases.

Signaling from the Rho family small GTPases controls a wide range of signaling outcomes. Key among the downstream effectors for many of the Rho GTPases are the p21-activated kinases, or PAK group. The PAK family comprises two types, the type I PAKs (PAK1, 2 and 3) and the type II PAKs (PAK4, 5 and 6), which have distinct structures and mechanisms of regulation.

Molecular basis for integrin adhesion receptor binding to p21-activated kinase 4 (PAK4).

Integrin adhesion receptors provide links between extracellular ligands and cytoplasmic signaling. Multiple kinases have been found to directly engage with integrin β tails, but the molecular basis for these interactions remain unknown. Here, we assess the interaction between the kinase domain of p21-activated kinase 4 (PAK4) and the cytoplasmic tail of integrin β5.

Allosteric MEK inhibitors act on BRAF/MEK complexes to block MEK activation.

The RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma.

Recognition of physiological phosphorylation sites by p21-activated kinase 4.

Many serine/threonine protein kinases discriminate between serine and threonine substrates as a filter to control signaling output. Among these, the p21-activated kinase (PAK) group strongly favors phosphorylation of Ser over Thr residues. PAK4, a group II PAK, almost exclusively phosphorylates its substrates on serine residues.

CDC42 binds PAK4 via an extended GTPase-effector interface.

The p21-activated kinase (PAK) group of serine/threonine kinases are downstream effectors of RHO GTPases and play important roles in regulation of the actin cytoskeleton, cell growth, survival, polarity, and development. Here we probe the interaction of the type II PAK, PAK4, with RHO GTPases. Using solution scattering we find that the full-length PAK4 heterodimer with CDC42 adopts primarily a compact organization.

Structural basis for Ufm1 recognition by UfSP.

Ubiquitin and ubiquitin-like proteins (Ubls) are involved in a variety of cellular functions, and dysfunction of these proteins often leads to disease, thus requiring the precise molecular recognition of the partner. Here, we report a structural basis for the recognition of Ufm1 by the Ufm1-specific protease (UfSP), both from Caenorhabditis elegans. Ufm1 functions in endoplasmic reticulum homeostasis, cell cycle regulation, and dysfunctions of this protein can result in breast cancer and neurological disorders.

The crystal structure of pseudokinase PEAK1 (Sugen kinase 269) reveals an unusual catalytic cleft and a novel mode of kinase fold dimerization.

The pseudokinase group encompasses some 10% of protein kinases, but pseudokinases diverge from canonical kinases in key motifs. The two members of the small new kinase family 3 (NKF3) group are considered pseudokinases. These proteins, pseudopodium-enriched atypical kinase 1 (PEAK1, Sugen kinase 269, or SgK269) and pragmin (Sugen kinase 223 or SgK223), act as scaffolds in growth factor signaling pathways, and both contain a kinase fold with degraded kinase motifs at their C termini.

PAK4 crystal structures suggest unusual kinase conformational movements.

Unlabelled: In order for protein kinases to exchange nucleotide they must open and close their catalytic cleft. These motions are associated with rotations of the N-lobe, predominantly around the 'hinge region'. We conducted an analysis of 28 crystal structures of the serine-threonine kinase, p21-activated kinase 4 (PAK4), including three newly determined structures in complex with staurosporine, FRAX486, and fasudil (HA-1077).

Structural and biochemical characterization of FabK from Thermotoga maritima.

TM0800 from Thermotoga maritima is one of the hypothetical proteins with unknown function. The crystal structure determined at 2.3 Å resolution reveals a two domain structure: the N-terminal domain forming a barrel and the C-terminal forming a lid.

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

The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs through its N-terminus, requiring both its Cdc42/Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency.

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

The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates.

Structure of the ABL2/ARG kinase in complex with dasatinib.

ABL2/ARG (ABL-related gene) belongs to the ABL (Abelson tyrosine-protein kinase) family of tyrosine kinases. ARG plays important roles in cell morphogenesis, motility, growth and survival, and many of these biological roles overlap with the cellular functions of the ABL kinase. Chronic myeloid leukemia (CML) is associated with constitutive ABL kinase activation resulting from fusion between parts of the breakpoint cluster region (BCR) and ABL1 genes.

Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity.

Eukaryotic 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.

Substrate and inhibitor specificity of the type II p21-activated kinase, PAK6.

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.

Structural alteration in the pore motif of the bacterial 20S proteasome homolog HslV leads to uncontrolled protein degradation.

In all cells, ATP-dependent proteases play central roles in the controlled degradation of short-lived regulatory or misfolded proteins. A hallmark of these enzymes is that proteolytic active sites are sequestered within a compartmentalized space, which is accessible to substrates only when they are fed into the cavity by protein-unfolding ATPases. HslVU is a prototype of such enzymes, consisting of the hexameric HslU ATPase and the dodecameric HslV protease.

RAC1P29S is a spontaneously activating cancer-associated GTPase.

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.

Type II p21-activated kinases (PAKs) are regulated by an autoinhibitory pseudosubstrate.

The type II p21-activated kinases (PAKs) are key effectors of RHO-family 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 full-length PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop.

Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma.

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.

Structure of ubiquitin-fold modifier 1-specific protease UfSP2.

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.