Granulovirus PK-1 kinase activity relies on a side-to-side dimerization mode centered on the regulatory αC helix

The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.

An evolutionary-conserved redox regulatory mechanism in human Ser/Thr protein kinases

ABSTRACTReactive oxygen species (ROS) are products of oxygen metabolism, but are also recognized as endogenous physiological mediators of cellular signaling. Eukaryotic protein kinase (ePK) regulation occurs through reversible phosphorylation events in the flexible activation segment. In this study, we demonstrate that the catalytic phosphotransferase output from the mitotic Ser/Thr kinase Aurora A is also controlled by cysteine (Cys) oxidation. Reversible regulation occurs by direct modification of a conserved residue (Cys 290), which lies adjacent to Thr 288, the activating site of phosphorylation. Strikingly, redox modulation of the Cys 290-equivalent in other ePKs is predicted to be an underappreciated regulatory mechanism, since ~100 human Ser/Thr kinases possess a Cys at this position in the conserved activation loop. Using real-time enzyme assays, we confirm that the presence of the equivalent Cys residue is prognostic for redox-sensitivity amongst a cohort of human CAMK, AGC and AGC-like kinases, including AKT, AMPK, CAMK1, MAPKAP-K2/3 and SIK1-3. Our findings demonstrate that dominant Cys-based redox-switching in the activation segment represents an evolutionary-conserved mode of regulation for a significant subset of the human kinome. This finding has important implications for understanding physiological and pathological signaling responses to ROS, and emphasises the importance of multivalent activation segment regulation in ePKs.ONE-SENTENCE SUMMARYThe catalytic activity of Ser/Thr kinases is regulated through a conserved Cys-based redox mechanism.

Screening of Microbial Extracts for Anticancer Compounds Using Streptomyces Kinase Inhibitor Assay

Eukaryotic kinases are known to play an important role in signal transduction pathways by phosphorylating their respective substrates. Abnormal phosphorylations by these kinases have resulted in diseases. Hence inhibitors of kinases are of considerable pharmaceutical interest for a wide variety of disease targets, especially cancers. A number of reports have been published which indicate that eukaryotic-like kinases may complement two-component kinase systems in several bacteria. In Streptomyces sp. such kinases have been found to have a role in formation of aerial hyphae, spores, pigmentation & even in antibiotic production in some strains. Eukaryotic kinase inhibitors are seen to inhibit formation of aerial mycelia in Streptomyces without inhibiting vegetative mycelia. This property has been used to design an assay to screen for eukaryotic kinase inhibitors. The assay involves testing of compounds against Streptomyces 85E ATCC 55824 using agar well diffusion method. Inhibitors of kinases give rise to “bald” colonies where aerial mycelia and sporulation inhibition is seen. The assay has been standardized using known eukaryotic protein kinase inhibiting anticancer agents like AG-490, AG-1295, AG-1478, Flavopiridol and Imatinib as positive controls, at a concentration ranging from 10 μg/well to 100 μg/well. Anti-infective compounds which are not reported to inhibit eukaryotic protein kinases were used as negative controls. A number of microbial cultures have been screened for novel eukaryotic protein kinase inhibitors. Further these microbial extracts were tested in various cancer cell lines like Panc1, HCT116, Calu1, ACHN and H460 at a concentration of 10 μg/mL/ well. The anticancer data was seen correlating well with the Streptomyces kinase assay thus validating the assay.