Protein phosphatase 2A (PP2A) is a major cellular serine/threonine protein phosphatase, present in the cell in a variety of heterotrimeric forms that differ in their associated regulatory B-subunit. Cloning of the mammalian B subunit has allowed the identification of a highly homologous Saccharomyces cerevisiae gene, RTS1. Disruption of the gene results in a temperaturesensitive growth defect that can be suppressed by expression of rabbit B ␣ or B ␥ isoforms. The B ␣ subunit is much more effective in restoring normal growth at 37°C than B ␥. Immunoprecipitated Rts1p was found associated with type 2A-specific protein phosphatase activity that is sensitive to 2 nM okadaic acid, but not to 100 nM phosphatase inhibitor-2, and to be phosphorylated in vivo. However, overexpression of RTS1 was unable to suppress the cold sensitivity, defective cytokinesis, and abnormal cell morphology resulting from defects in the CDC55 gene, which encodes the yeast homolog of a different B subunit of another form of 2A phosphatase, PP2A 1 . These results indicate that Rts1p is a yeast homolog of the mammalian B subunit and that the various regulatory B-subunits of PP2A are not functionally redundant but direct the enzyme to distinct cellular functions.

The yeast gene PBS2 encodes a presumed protein kinase. The gene is essential for manifestation of resistance to the antibiotic polymyxin B. Deletion of PBS2 enables a ras2-530 null mutant to grow on nonfermentable carbon sources; overexpression of PBS2+ enhances viability of a mutant. Overexpression of PBS2+ also diminishes cellular response to mating pheromone MFa. These results suggest that the PBS2 and RAS2 genes affect a common pathway that may communicate with the pheromone response pathway. In addition, disruption of PBS2 renders cells sensitive to high osmolarity: exposure to 0.9 M-Naa causes growth arrest, appearance of bizarre morphological forms, and eventual death. A mutation suppressing pbs2 deletion has been found. That mutation restores full polymyxin B resistance but only partially corrects the osmotic sensitivity defect. These observations indicate that PBS2 is involved in diverse physiological pathways in yeast.

Polymyxin B is an antibiotic that kills sensitive cells by disrupting their membranes. We have cloned a wild-type yeast gene that, when present on a high-copy-number plasmid, renders the cells resistant to the drug. The nucleotide sequence of this gene is presented. A single open reading frame within the sequence has the potential to encode a polypeptide (molecular mass of 77.5 kDa) that shows strong homologies to polypeptides of the protein kinase family. The gene, PBS2, located on 'chronmosome X, is not allelic to the previously described PBS) gene (where PBS signifies polymyxin B sensitivity). Although pbs) mutations confer resistance to high levels of polymyxin B, double mutants, pbs) pbs2, are not resistant to the drug, indicating that PBS2 is essential for pbs) activity.Models based on the proposed protein kinase activity of the PBS2 gene product are presented to explain the interaction between PBSI and PBS2 gene products involved in conferring polymyxin B resistance on yeast cells.

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)-and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulinlike growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKAdependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.

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