The inhibition of protein phosphorylation is proving to be a powerful therapeutic approach to treat a number of disorders. As more protein kinase inhibitors are developed, critical issues of drug and target specificity must be addressed. While a remarkable amount of information exists for the regulation of protein kinase function, relatively little is known about the substrates they phosphorylate. Classical protein kinase substrate identification approaches have been hampered largely due to high background phosphorylation in cellular lysates and the laborious methods required identifying and purifying both the protein kinases and their substrates. Recent advances in biochemistry, bioinformatics and proteomics have addressed many of these issues. A particularly powerful method to identify direct protein kinase substrates combines standard proteomics with a unique chemical genetics approach. Identification of new protein kinase substrates by such chemical-genetic and other emerging technologies may provide the next generation of drug targets for multiple therapeutic areas. Keywords: chemical genetics, analog sensitive kinase alleles, phosphorylation, functional proteomics, mass spectrometry

The chemokine receptor CXCR4 is a co-receptor for T-tropic strains of HIV-1. A number of small molecule antagonists of CXCR4 are in development but all are likely to lead to adverse effects due to the physiological function of CXCR4. To prevent these complications, allosteric agonists may be therapeutically useful as adjuvant therapy in combination with small molecule antagonists. A synthetic cDNA library coding for 160,000 different SDF-based peptides was screened for CXCR4 agonist activity in a yeast strain expressing a functional receptor. Peptides that activated CXCR4 in an autocrine manner induced colony formation. Two peptides, designated RSVM and ASLW, were identified as novel agonists that are insensitive to the CXCR4 antagonist AMD3100. In chemotaxis assays using the acute lymphoblastic leukemia cell line CCRF-CEM, RSVM behaves as a partial agonist and ASLW as a superagonist. The superagonist activity of ASLW may be related to its inability to induce receptor internalization. In CCRF-CEM cells, the two peptides are also not inhibited by another CXCR4 antagonist, T140, or the neutralizing monoclonal antibodies 12G5 and 44717.111. These results suggest that alternative agonist-binding sites are present on CXCR4 that could be screened to develop molecules for therapeutic use.

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G(s)alpha. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C(1) and C(2)) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the beta4-beta5 loop of the C(2) domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C(1) domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C(2) domain of adenylyl cyclase for the wild type C(1) domain was exploited to isolate a complex containing VC(1), IIC(2), and G(s)alpha-guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in the absence of forskolin and a complex of VC(1), IIC(2), forskolin, and P-site inhibitor in the absence of G(s)alpha-GTPgammaS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G(s)alpha.

Yeasts have three functionally redundant G1 cyclins required for cell cycle progression through G1. Mutations in GIN4 and CLA4 were isolated in a screen for mutants that are inviable with deletions in the G1 cyclins CLN1 and CLN2. cln1 cln2 cla4 and cln1 cln2 gin4 cells arrest with a cytokinesis defect; this defect was efficiently rescued by CLN1 or CLN2 expression. GIN4 encodes a protein with strong homology to the Snflp serine/threonine kinase. Cla4p is homologous to mammalian p21-activated kinases (PAKs) (kinases activated by the rho-class GTPase Rac or Cdc42). We developed a kinase assay for Cla4p. Cla4p kinase was activated in vivo by the GTP-bound form of Cdc42p. The specific activity of Cla4p was cell cycle regulated, peaking near mitosis. Deletion of the Cla4p pleckstrin domain diminished kinase activity nearly threefold and eliminated in vivo activity. Deletion of the Cla4p Cdc42-binding domain increased kinase activity nearly threefold, but the mutant only weakly rescued cla4 function in vivo. This suggests that kinase activity alone is not sufficient for full function in vivo. Deletion of the Cdc42-binding domain also altered the cell cycle regulation of kinase activity. Instead of peaking at mitosis, the mutant kinase activity exhibited reduced cell cycle regulation and peaked at the G1/S border. Cla4p kinase activity was not reduced by mutational inactivation of gin4, suggesting that Gin4p may be downstream or parallel to Cla4p in the regulation of cytokinesis.

In budding yeast, one of three G1 cyclins is required for progression though START, when cells commit to a further round of cell division. We have identified mutations in ALG1 (ERC14), a gene required for N-glycosylation, which are inviable in a cln1 cln2 background but are rescued by over-expression of CLNs. CLN1 and CLN2 are much more efficient than CLN3 in rescuing the erc14-1 allele. The erc14-1 allele results in a significant N-glycosylation defect, and no rescue of this defect by CLN1 over-expression was detected. These data suggest that CLN over-expression could be allowing cells to live with lower levels of N-glycosylation, possibly by overcoming a checkpoint sensitive to N-glycosylation capacity. A plasmid suppressor of alg1, PSA1, encodes a 361 amino-acid protein with homology to NDP-hexose pyrophosphorylases, the enzymes that catalyze the formation of activated sugar nucleotides. PSA1 is an essential gene, and PSA1 transcription is nearly co-ordinately regulated with CLN2 transcription, peaking near START. Co-ordinate regulation of glycosylation, sugar nucleotide metabolism, and cell-cycle progression through G1 may be a feature that ensures adequate cell-wall precursors are present before bud emergence.