sck1, a high copy number suppressor of defects in the cAMP-dependent protein kinase pathway in fission yeast, encodes a protein homologous to the Saccharomyces cerevisiae SCH9 kinase.

Schizosaccharomyces pombe regulates intracellular cAMP levels, and thus cAMP-dependent protein kinase (PKA) activity, in response to changes in nutrient conditions. Mutations in any of eight git genes inhibit glucose repression of fbp1 transcription, alter the cell morphology, and cause a reduction in the growth rate. The eight git genes encode components of an adenylate cyclase activation pathway, adenylate cyclase itself, and the catalytic subunit of PKA. Three of these genes have been identified in other studies as regulators of meiosis. Here we show that the sck1 gene, cloned as a high copy number suppressor of a mutation in git3, is able to suppress the defects conferred by a mutation in any of these git genes. Sequence analysis suggests that sck1 encodes a protein most closely related to the Saccharomyces cerevisiae SCH9 protein kinase that had previously been identified as a high copy number suppressor of mutations in S. cerevisiae that reduce or eliminate PKA activity. Disruption of the sck1 gene causes a significant delay in exit from stationary phase when combined with a disruption of the pka1 (git6) gene encoding the catalytic subunit of PKA. However, the sck1 disruption by itself has little or no effect upon fbp1 transcription, meiosis, or exit from stationary phase, and does not enhance the constitutive fbp1 transcription observed in a pka1 mutant. Therefore, sck1 appears to function in a redundant fashion to pka1, but to varying degrees, in the pathways regulated by pka1.

Regulation of integrin affinity states through an NPXY motif in the beta subunit cytoplasmic domain.

The ligand binding affinities of the integrins are regulated through their cytoplasmic domains. To identify specific residues that are involved in this process, we have generated mutants in the beta 1 and beta 3 tails and coexpressed them in Chinese hamster ovary cells with constitutively active alpha subunits. These alpha subunits are chimera of extra-cellular and transmembrane alpha IIb joined to the cytoplasmic domains of alpha 5, alpha 6A, or alpha 6B and confer an energy-dependent high affinity state when expressed in Chinese hamster ovary cells. The affinity state of these transfectants was determined by analyzing the binding of PAC1, an antibody that specifically recognizes the activated form of the reporter group, extracellular alpha IIb beta 3. We have identified point mutants in several areas of the beta tails, which result in a reduced ability to bind ligand. Complete abolition of PAC1 binding was obtained with mutants in an NPXY motif found in many integrin beta subunits and implicated in the internalization of other cell surface receptors. Similar effects on PAC1 binding were observed whether coexpression was with alpha chimera containing alpha 5, alpha 6A, or alpha 6B cytoplasmic sequences. These studies identify a novel role for the NPXY motif in the regulation of integrin binding affinity.