Kaposi's sarcoma-associated herpesvirus K-bZIP protein is phosphorylated by cyclin-dependent kinases.

The K8 locus in Kaposi's sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three alternatively spliced transcripts. The fully spliced transcript encodes K-bZIP, the KSHV homologue of the EBV immediate-early transcriptional transactivator Z. Here we show that despite the presence of alternatively spliced transcripts, the protein from the fully spliced RNA, K-bZIP, is the principal product detectable in KSHV-infected B cells. The protein is detected only in lytically infected cells and is localized to the nucleus. We further characterized K-bZIP by determining its phosphorylation status. Phosphoamino acid analysis revealed phosphorylation on serine and threonine. Analysis of the sites of K-bZIP phosphorylation by tandem mass spectrometry revealed that K-bZIP was phosphorylated on Thr 111 and Ser 167. These phosphorylation sites are contained within cyclin-dependent kinase (CDK) recognition sites with the consensus sequence (S/T)PXR, suggesting that K-bZIP could be phosphorylated by CDKs. We tested this hypothesis using an in vitro kinase reaction performed in whole-cell extracts that resemble in vivo conditions more closely than standard in vitro kinase reactions. We found that the three CDK-cyclin complexes we tested phosphorylated K-bZIP but not the control ORF 73 protein, which contains four (S/T)PXR sites. Ectopic expression of K-bZIP cannot reactivate KSHV from latency, and single and double mutants of K-bZIP in which alanines replaced the phosphorylated serine and/or threonine also failed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the protein.

A chemical switch for inhibitor-sensitive alleles of any protein kinase.

Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.