We have catalogued the protein kinase complement of the human genome (the "kinome") using public and proprietary genomic, complementary DNA, and expressed sequence tag (EST) sequences. This provides a starting point for comprehensive analysis of protein phosphorylation in normal and disease states, as well as a detailed view of the current state of human genome analysis through a focus on one large gene family. We identify 518 putative protein kinase genes, of which 71 have not previously been reported or described as kinases, and we extend or correct the protein sequences of 56 more kinases. New genes include members of well-studied families as well as previously unidentified families, some of which are conserved in model organisms. Classification and comparison with model organism kinomes identified orthologous groups and highlighted expansions specific to human and other lineages. We also identified 106 protein kinase pseudogenes. Chromosomal mapping revealed several small clusters of kinase genes and revealed that 244 kinases map to disease loci or cancer amplicons.

The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N-and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N-and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics.Newly synthesized Ras proteins are partitioned to the cytoplasmic face of the plasma membrane by a series of posttranslational modifications. The first step, catalyzed by the enzyme farnesyl protein transferase, is the addition of the 15-carbon isoprenyl group farnesyl to the sulfhydryl group of cysteine in the Ras carboxyl-terminal CAAX box (where C is cysteine, A is aliphatic, and X is typically Met or Ser) (1-3). Farnesylation is followed by proteolytic removal of the AAX amino acids and methylation of the carboxyl group of the farnesylated cysteine (4). Ras proteins at the plasma membrane cycle between an active GTP-bound state and an inactive GDPbound state. Mutations that stabilize the active GTP-bound state have been identified in over 30% of human tumors, with particularly high incidences in pancreatic (ϳ90%) and colon (ϳ50%) cancers. Four oncogenic Ras proteins have been described, H-Ras, N-Ras, K-Ras4A, and K-Ras4B. The majority of mutations associated with human cancer have been found in the K-Ras gene. The two K-Ras proteins are products of a single alternatively spliced transcript, with K-Ras4B the predominant isoform (Ͼ80%) (5, 6).Ras proteins that have been genetically modified so that they lack the isoprenylated cysteine do not associate with the plasma membrane and cannot transform fibroblasts (7). These genetic experiments provided the basis for the development of farnesyl transferase inhibitors (FTIs) 1 as anti-cancer agents. A number of reports have demonstrated that pharmacological inhibition of farnesyl protein transferase by CAAX analogs reduces anchorage-independent growth of Ras-transformed cells in soft agar (8) and slows growth of Ras-transformed cells in nude mice (9, 10). The FTIs appear relatively non-toxic in that they do not interfere with normal cell proliferation (11). This result was somewhat surprising because Ras function was shown to be necessary for normal growth factor signaling and cell proliferation (12). A mechanism through which cells may proliferate in...

Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth.

Caenorhabditis elegans should soon be the first multicellular organism whose complete genomic sequence has been determined. This achievement provides a unique opportunity for a comprehensive assessment of the signal transduction molecules required for the existence of a multicellular animal. Although the worm C. elegans may not much resemble humans, the molecules that regulate signal transduction in these two organisms prove to be quite similar. We focus here on the content and diversity of protein kinases present in worms, together with an assessment of other classes of proteins that regulate protein phosphorylation. By systematic analysis of the 19,099 predicted C. elegans proteins, and thorough analysis of the finished and unfinished genomic sequences, we have identified 411 full length protein kinases and 21 partial kinase fragments. We also describe 82 additional proteins that are predicted to be structurally similar to conventional protein kinases even though they share minimal primary sequence identity. Finally, the richness of phosphorylation-dependent signaling pathways in worms is further supported with the identification of 185 protein phosphatases and 128 phosphoprotein-binding domains (SH2, PTB, STYX, SBF, 14-3-3, FHA, and WW) in the worm genome. R eversible protein phosphorylation plays a central role in regulating basic functions of all eukaryotes such as DNA replication, cell cycle control, gene transcription, protein translation, and energy metabolism. Protein phosphorylation is also required for more advanced functions in higher eukaryotes such as cell, organ, and limb differentiation, cell survival, synaptic transmission, cell-substratum and cell-cell communication, and to mediate complex interactions with the external environment. Because aberrant protein phosphorylation is commonly the cause of cancer and other human diseases, a comprehensive knowledge of the key enzymes that regulate these functions can provide the basis for novel therapeutic intervention strategies.The genomic revolution promises to provide a new paradigm for drug discovery, allowing one to selectively target the molecular basis of human disease. The completion of the Caenorhabditis elegans genome sequence gives us an opportunity to decipher the molecular nature of its signal transduction machinery. Several global analyses of proteins and protein domains present in C. elegans have been presented elsewhere (1-4), revealing that protein kinases comprise the second largest family of protein domains in worms. The three most frequently occurring protein domains found in worms are seven transmembrane chemoreceptors (650 domains, 3.5% of genome), protein kinases (496 domains, 2.6% of genome), and zinc finger C4 domains, including nuclear hormone receptors (275 domains, 1.4% of genome). A more in-depth analysis has been performed on the 535 worm proteins containing zinc-binding domains, including the C4, C2H2, and C3HC4 ring finger types (3), and on the 83 worm homeobox transcription factors (4). Here, we present a comparative ana...

HGK (hepatocyte progenitor kinase-like/germinal center kinase-like kinase) is a member of the human STE20/mitogen-activated protein kinase kinase kinase kinase family of serine/threonine kinases and is the ortholog of mouse NIK (Nck-interacting kinase). We have cloned a novel splice variant of HGK from a human tumor line and have further identified a complex family of HGK splice variants. We showed HGK to be highly expressed in most tumor cell lines relative to normal tissue. An active role for this kinase in transformation was suggested by an inhibition of H-Ras V12 -induced focus formation by expression of inactive, dominantnegative mutants of HGK in both fibroblast and epithelial cell lines. Expression of an inactive mutant of HGK also inhibited the anchorage-independent growth of cells yet had no effect on proliferation in monolayer culture. Expression of HGK mutants modulated integrin receptor expression and had a striking effect on hepatocyte growth factor-stimulated epithelial cell invasion. Together, these results suggest an important role for HGK in cell transformation and invasiveness.The mammalian STE20/mitogen-activated protein kinase kinase kinase kinase (MAP4K) family consists of 28 serine/threonine kinases related in their catalytic domains (reviewed in reference 14). By analogy with the prototype STE20 kinase in Saccharomyces cerevisiae, mammalian MAP4K kinases are likely to regulate changes in transcription, cytoskeletal organization, and cell cycle progression in response to extracellular signals (17). Comparison of the overall domain structure places these kinases into two structural classes, the p21-activated protein kinases (PAKs) (1) and germinal center kinases (GCKs) (28). The GCK kinases lack the regulatory Cdc42/ Rac-interacting domain found in the PAKs, having instead an N-terminal kinase domain and a C-terminal extension of variable length. Unlike PAKs, several GCKs appear to be activated in the absence of stimuli when overexpressed (3,10,38), suggesting that these kinases are regulated either by oligomerization or by binding of negative regulatory factors.GCK kinases show little sequence homology outside of the kinase domain and are further broken down into nine subfamilies (14, 31a). HGK (hepatocyte progenitor kinase-like/GCKlike kinase) is one of four members of the GCK group IV (recently renamed the MSN subfamily) that also includes TNIK, MINK, and NRK/NESK (13,22,27,35,51). HGK, TNIK, and MINK are highly homologous in their kinase and C-terminal domains (about 92 and 87% amino acid identity, respectively), with variability in the intervening region that is less conserved (53% between HGK and TNIK). NRK/NESK is more divergent, with only 59% homology in the kinase domain and 37% homology in the C-terminal domain. The C-terminal domain is a citron homology (CNH) domain, named for citron rho-interacting kinase (CRIK), where it was first described (16,31). CNH domains are found not only in the GCK group IV/MSN kinases but also in group I GCKs (KHS subfamily) (14, 31a), as well as in pr...