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Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.

ABSTRACT: Recent studies have shown that there exists a family of protein kinases structurally and functionally related to the yeast cell cycle regulatory kinase cdc2 [Meyerson, M., Faha, B., Su, L.-K., Harlow, E. & Tsai, L.-H. (1991) Cold Spring Harbor Symp. Quant. Biol. 56, 177-186 and Meyerson, M., Enders, G. H., Wu, C.-L., Su, L.-K., Gorka, C., Nelson, C., Harlow, E. & Tsai, L.-H. (1992) EMBO J. 11, 2909-2917]. Two members of cdc2 family, p34cdc2 (also named cdk1) and cdk2, have been identified in mammalian cells. cdk1 kinase regulates the progression from G2 to M phase, and cdk2 kinase has been proposed to regulate the progression from G1 to S phase. In this work, we have cloned and structurally characterized a third member of the cdc2 kinase family with 58% amino acid sequence identity to mouse cdk1 and 61% identity to human cdk2. We call this kinase neuronal cdc2-like kinase (nclk) because, in contrast to either cdk1 or cdk2, nclk is expressed at high levels in terminally differentiated neurons no longer in the cell cycle. Previous studies have shown [Hisanaga, S., Kusubata, M., Okumura, E. & Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803 and Guan, R. J., Hall, F. L. & Cohlberg, J. A. (1992) J. Neurochem. 58, 1365-1371] that cdk1 kinase, but not other structurally defined protein kinases, could phosphorylate the repeated Lys-Ser-Pro (KSP) motifs found in mammalian high and middle molecular mass neurofilament subunits in vitro, but the precise molecular nature of the endogenous neuronal KSP kinase has remained undefined. The structural similarity of nclk to cdk1 kinase and its high level of expression in terminally differentiated neurons suggest that nclk may play a role in the phosphorylation of the neurofilament KSP repeats in vivo, a function distinct from cell cycle regulation.

SUBMITTER: Hellmich MR

PROVIDER: S-EPMC50443 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.

Hellmich M R MR Pant H C HC Wada E E Battey J F JF

Proceedings of the National Academy of Sciences of the United States of America 19921101 22

Recent studies have shown that there exists a family of protein kinases structurally and functionally related to the yeast cell cycle regulatory kinase cdc2 [Meyerson, M., Faha, B., Su, L.-K., Harlow, E. & Tsai, L.-H. (1991) Cold Spring Harbor Symp. Quant. Biol. 56, 177-186 and Meyerson, M., Enders, G. H., Wu, C.-L., Su, L.-K., Gorka, C., Nelson, C., Harlow, E. & Tsai, L.-H. (1992) EMBO J. 11, 2909-2917]. Two members of cdc2 family, p34cdc2 (also named cdk1) and cdk2, have been identified in mam ...[more]

PMID: 1279696

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Project description:BACKGROUND: In search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery. METHODS: A new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm. RESULTS: We have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 ??. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial kinases was subsequently undertaken to explain and rationalize the selectivity trend determined by the in vitro binding assays. Interestingly, the latter analysis showed that selectivity could be correlated with differences in kinase solvation thermo dynamics induced by minor sequence variations of the otherwise highly similar ATP binding pockets. CONCLUSIONS: In conclusion, 3'-bulky amino substituted 6-BIO derivatives, which demonstrate enhanced specificity towards LGSK-3, represent a new scaffold for targeted drug development to treat leishmaniasis.

Dataset Information

Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.

Publications

Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.

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