Mitogen-activated protein (MAP) kinases, also known as extracellular signal-regulated kinases (ERKs), are thought to act at an integration point for multiple biochemical signals because they are activated by a wide variety of extracellular signals, rapidly phosphorylated on threonine and tyrosine, and highly conserved. A critical protein kinase lies upstream of MAP kinase and stimulates the enzymatic activity of MAP kinase. The structure of this protein kinase, denoted MEK1, for MAP kinase or ERK kinase, was elucidated from a complementary DNA sequence and shown to be a protein of 393 amino acids (43,500 daltons) that is related most closely in size and sequence to the product encoded by the Schizosaccharomyces pombe byr1 gene. The MEK gene was highly expressed in murine brain, and the product expressed in bacteria phosphorylated the ERK gene product.

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Elevated expression of mammalian polo-like kinase (Plk)1 occurs in many different types of cancers, and Plk1 has been proposed as a novel diagnostic marker for several tumors. We used the recently developed vector-based small interfering RNA technique to specifically deplete Plk1 in cancer cells. We found that Plk1 depletion dramatically inhibited cell proliferation, decreased viability, and resulted in cell-cycle arrest with 4 N DNA content. The formation of dumbbell-like chromatin structure suggests the inability of these cells to completely separate the sister chromatids at the onset of anaphase. Plk1 depletion induced apoptosis, as indicated by the appearance of subgenomic DNA in fluorescence-activated cellsorter (FACS) profiles, the activation of caspase 3, and the formation of fragmented nuclei. Plk1-depletion-induced apoptosis was partially reversed by cotransfection of nondegradable mouse Plk1 constructs. In addition, the p53 pathway was shown to be involved in Plk1-depletion-induced apoptosis. DNA damage occurred in Plk1-depleted cells and inhibition of ATM strongly potentiated the lethality of Plk1 depletion. Although p53 is stabilized in Plk1-depleted cells, DNA damage also occurs in p53 ؊/؊ cells. These data support the notion that disruption of Plk1 function could be an important application in cancer therapy. T he polo kinase family includes mammalian polo-like kinase (Plk)1, Snk, Fnk, Xenopus laevis Plx1, Drosophila polo, fission yeast Plo1, and budding yeast Cdc5 (1). Genetic and biochemical experiments in various organisms indicate that polo-like kinases are important regulators of many cell-cycle-related events, including activation of Cdc2, chromosome segregation, centrosome maturation, bipolar spindle formation, regulation of anaphase-promoting complex, and execution of cytokinesis (1, 2).To investigate the functions of Plk1 in mammalian cells, we previously directly transfected 21-nucleotide double-stranded RNA into the cells to deplete Plk1 (3). We found that Plk1 depletion results in elevated Cdc2 protein kinase activity and thus attenuates cell-cycle progression. About 45% of cells treated with Plk1 small interfering RNA (siRNA) show the formation of a dumbbell-like DNA organization, suggesting that sister chromatids are not completely separated. About 15% of these cells do complete anaphase but do not complete cytokinesis. Finally, Plk1 depletion significantly reduces centrosome amplification in hydroxyurea-treated U2OS cells (3).A close correlation between mammalian Plk1 expression and carcinogenesis was recently documented. Mammalian Plk1 was found to be overexpressed in various human tumors, including head and neck squamous cell carcinomas, oropharyngeal carcinomas, non-small cell lung cancer, melanomas, and ovarian and endometrial carcinomas (4). It was proposed that Plk1 could be used as a novel diagnostic marker for several types of cancers (4-6). Furthermore, constitutive expression of Plk1 in NIH 3T3 cells causes oncogenic focus formation and induces tumor growth in nude mice (7)...

Incorporation of phosphorus from [y-30PJATP into protein was catalyzed by specific immunoprecipitates from avian sarcoma virus (ASV-transformed avian and mammalian cells. This incorporation was observed only when antiserum from tumor-bearing rabbits able to specifically precipitate the ASV sarcoma gene product, p~o srC was used to immunoprecipitate antigens from transformed cell lysates. Immunoprecipitates of extracts from normal cells or cells infected with a transformation-defective ASV mutant showed no activity in this assay, nor did any immune complexes formed with normal rabbit serum and any of the cell extracts tested. The expression of the protein-kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was growth temperature-dependent in cells infected with an ASV mutant temperature-sensitive for transformation. These results on an enzymatic activity associated with the ASV transforming protein are discussed in terms of protein phosphorylation as a mechanism for viral transformation.Four genes have been identified and mapped on the avian sarcoma virus (ASV) genome (1, 2). Three of these genes-gag, pol, and env-code for the virion structural proteins, including the group-specific (gs) viral core proteins, the RNA-directed DNA polymerase, and the envelope glycoproteins, respectively. The fourth gene, designated src, has been identified through the isolation of temperature-sensitive (ts) and deletion mutants defective in transformation in vitro and sarcoma induction in vivo (3-7).Recent work from this laboratory has resulted in the identification of a protein of molecular weight (Mr) 60,000 that appears to be the product of the ASV src gene (8-10). Determination that this protein is actually the product of the src gene is based on the following data: (i) It was detected as a nonstructural, transformation-specific antigen in ASV-transforned chicken cells, ASV-transformed mammalian cells, and ASVinduced mammalian tumor cells, by immunoprecipitation of radiolabeled cell extracts with serum from ASV-tumor-bearing rabbits. (ii) In vitro cell-free translation of the 3' third of nondefective ASV viral RNA, the region of the genome that contains the src gene, resulted in the synthesis of a polypeptide of Mr 60,000. (iii) The polypeptide of Mr 60,000 made in vitro by cell-free translation and the transformation-specific antigen isolated by immunoprecipitation of all types of ASV-infected cell extracts tested are identical as determined by peptide analyses (ref. 10; J. S. Brugge, E. Erikson, M. S. Collett, and R. L. Erikson, unpublished results). We feel that it is consistent with these data to conclude that the protein of Mr 60,000 is the product of the ASV src gene and consequently give it the following designation: p60src.In order to further elucidate the mechanism of ASV-induced Preparation of Antiserum and Immunoprecipitation. Antiserum was obtained from New Zealand rabbits in which tumors had been induced by injection of purified SR-ASV as previously described (8, 13). Several preparations of antis...

Significance Found in most eukaryotic cells, a centriole is a cylindrically shaped subcellular structure that plays an important role in various cellular processes, including mitotic spindle formation and chromosome segregation. Centriole duplication occurs only once per cell cycle, thus ensuring accurate control of centriole numbers to maintain genomic integrity. Although a growing body of evidence suggests that a Ser/Thr protein kinase, polo-like kinase 4 (Plk4), is a key regulator of centriole duplication, how Plk4 is recruited to centrosomes remains largely unknown. Here we showed that Plk4 dynamically localizes to distinct subcentrosomal regions by interacting with two hierarchically regulated scaffolds, Cep192 and Cep152. Highlighting the importance of these interactions, mutational disruption of either one of these interactions was sufficient to cripple Plk4-dependent centriole biogenesis.