. Cell. Biol. 8: [4981][4982][4983][4984][4985][4986][4987][4988][4989][4990] 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987 (8,13). The beta subunit is required for maximal activity of the catalytic subunit (10) and becomes phosphorylated when the holoenzyme is allowed to undergo autophosphorylation. Although the beta subunit presumably plays a regulatory role, neither the function of this polypeptide nor the significance of autophosphorylation is well understood. The recognition site for casein kinase II consists of a serine or threonine residue located immediately N terminal to a cluster of acidic amino acids (22,25

2-aminothiazole (1) was discovered as a novel Src family kinase inhibitor template through screening of our internal compound collection. Optimization through successive structure-activity relationship iterations identified analogs 2 (Dasatinib, BMS-354825) and 12m as pan-Src inhibitors with nanomolar to subnanomolar potencies in biochemical and cellular assays. Molecular modeling was used to construct a putative binding model for Lck inhibition by this class of compounds. The framework of key hydrogen-bond interactions proposed by this model was in agreement with the subsequent, published crystal structure of 2 bound to structurally similar Abl kinase. The oral efficacy of this class of inhibitors was demonstrated with 12m in inhibiting the proinflammatory cytokine IL-2 ex vivo in mice (ED50 approximately 5 mg/kg) and in reducing TNF levels in an acute murine model of inflammation (90% inhibition in LPS-induced TNFalpha production when dosed orally at 60 mg/kg, 2 h prior to LPS administration). The oral efficacy of 12m was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally at 0.3 and 3 mg/kg twice daily. Dasatinib (2) is currently in clinical trials for the treatment of chronic myelogenous leukemia.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase H have been isolated by immunological screening of Agtll expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinases suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.Casein kinase II is a cyclic nucleotide-independent, Ca2l-and calmodulin-insensitive protein kinase which is widely distributed among eucaryotic organisms (15). The enzyme phosphorylates a broad spectrum of both nuclear and nonnuclear substrates (1,5,11,15), suggesting that it may function in the regulation or integration of cell metabolism. Casein kinase II from most sources is composed of two dissimilar subunits, alpha (35 to 44 kilodaltons [kDa]) and beta (24 to 29 kDa), which combine to form a native 02f2 tetramer with a molecular weight of 130,000 to 150,000. The enzyme can utilize either ATP or GTP as the nucleoside triphosphate donor and phosphorylates serine or threonine residues in protein substrates. A cluster of acidic residues located immediately C-terminal to the modified residue appears to be important for substrate recognition (15, 25). The alpha polypeptide has been identified as the catalytic subunit by three independent methods (6,17,26). The beta subunit becomes phosphorylated when the enzyme is allowed to undergo autophosphorylation, but the function of this subunit and the significance of autophosphorylation are not known.In an effort to initiate a genetic analysis of casein kinase II, we previously isolated and characterized the enzyme from Drosophila melanogaster (14). The purified kinase is composed of a 37-kDa alpha and 28-kDa beta subunit which form a 130,000-dalton a232 tetramer. Like its counterpart in other species, the enzyme phosphorylates both serine and threonine residues, utilizes either ATP or GTP, exhibits autophosphorylation of the beta subunit, and is strongly inhibited by heparin, a characteristic feature of casein kinase II (15). In addition, the Drosophila enzyme displays a protein substrate specificity which is virtually indistinguishable from that exhibited by the enzyme from calf thymus (7). Peptide mapping and immunological studies demonstrate that the insect and mammalian enzymes are homologous and suggest that casein kinase II has been ...

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1⌬, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2⌬ cells exhibit morphological defects. At permissive temperatures, rom2⌬ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1⌬ and kar3⌬ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2⌬ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton. INTRODUCTIONThe cytoskeleton is the infrastructure of the cell; it aids in determining cell shape and participates in many dynamic cellular processes. Two major components of all eukaryotic cytoskeletons are microfilaments, composed of actin subunits, and microtubules, made up of tubulin heterodimers. The two systems are involved in distinct processes within the cell. This is particularly evident in the budding yeast Saccharomyces cerevisiae. The primary functions of the yeast actin cytoskeleton are in secretion, cell growth and polarized morphogenesis, resulting in bud and mating projection formation (Adams and Pringle, 1984;Kilmartin and Adams, 1984;Novick and Botstein, 1985;Read et al., 1992). Yeast microtubules are dispensable for secretion and cell growth; instead, they are required for nuclear positioning, chromosome segregation during mitosis and meiosis, and nuclear fusion during mating (karyogamy; Adams and Pringle, 1984;Kilmartin and Adams, 1984;Huffaker et al., 1988;Jacobs et al., 1988;Snyder et al., 1991; reviewed in Page and Snyder, 1993).Studies of the regulation of cytoskeletal function and rearrangement in a wide variety of eukaryotes have provided insight into the mechanisms controlling the organization of microfilaments and, to a lesser degree, microtubules. A family of highly conserved ras-homologous small GTPases, known as Rho proteins, have emerged as key molecular switches that regulate organization of the actin cytoskeleton (reviewed in Hall, 1994;Ridley, 1995).Rho proteins cycle between a GTP-bound active state and a GDP-bound inactive state with the assistance of several important classes of regulatory proteins. One type of Rho regulator is the GDP/GTPexchange factor (GEF), which stimulates release of GDP from the Rho protein, allowing subsequent binding of GTP and thereby Rho activation (...

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which must be encoded by separate genes (R. Padmanabha and C. V. C. Glover, J. Biol. Chem. 262:1829Chem. 262: -1835Chem. 262: , 1987 Casein kinase II is a cyclic-nucleotide-independent protein kinase which phosphorylates a broad spectrum of both cytoplasmic and nuclear substrates (for a review, see reference 10). The enzyme is widely distributed among eucaryotic organisms and has been purified from a number of mammalian and avian species (10), Drosophila melanogaster (8), and Saccharomyces cerevisiae (19,22 (12,26), and in some systems two related alphalike polypeptides, alpha and alpha', have been observed (4, 10). The beta subunit becomes phosphorylated when the enzyme is allowed to undergo autophosphorylation, but the function of this subunit and the significance of autophosphorylation are poorly understood. The enzyme phosphorylates Ser and Thr residues in protein substrates, and a cluster of acidic residues immediately C-terminal to the modified residue appears to be important for recognition (15,18,20). The properties of casein kinase II have been highly conserved over large evolutionary distances (10,22,26), implying that the enzyme fulfills an important functional role.The purified yeast enzyme contains two related catalytic subunits (alpha, 42 kDa; alpha', 35 kDa), a beta subunit of 41 kDa, and possibly also a beta' subunit of 32 kDa (22 (17). JM105 was grown in 2x YT medium (2x YT is 1.6% tryptone, 1% yeast extract, and 0.5% NaCl) and plated on H plates (1% tryptone, 0.8% NaCl, and 1.2% Bacto-Agar).