Search

Your search keyword '"Mittnacht S"' showing total 5 results
Start Over Author "Mittnacht S" Journal journal of biological chemistry
5 results on '"Mittnacht S"'

1. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases.

Author

Zarkowska, T and Mittnacht, S

Abstract

The retinoblastoma tumor suppressor protein, pRB, is inactivated by phosphorylation. While existing evidence is strong that such phosphorylation is mediated by one or more cyclin-dependent kinases (CDKs) active during G1/S, it remains unclear which of the various CDKs is responsible. We show here that three candidate pRB-inactivating kinases, CDK4-cyclin D1, CDK2-cyclin E, and CDK2-cyclin A, phosphorylate pRB differentially, each on a subset of authentic pRB phosphorylation sites. Notably, two neighboring pRB phosphate acceptors, threonine 821 and threonine 826, which have previously been implicated in the regulation of LXCXE protein binding, are phosphorylated by different CDKs. We demonstrate that phosphorylation by either CDK2-cyclin A, which phosphorylates T821, or CDK4-cyclin D1, which phosphorylates threonine 826, can disable pRB for subsequent binding of an LXCXE protein. However, only one of these two kinases, CDK2-cyclin A, can dissociate a pre-existing LXCXE protein-pRB complex. We provide evidence that prior binding of an LXCXE protein blocks access to certain residues specifically targeted by CDK4-cyclin D1, explaining the inability of this kinase to resolve such complexes. While these results are not direct proof of the relevance of differential pRB phosphorylation in cells, our findings support a model whereby full phosphorylation of pRB may require the action of more than one kinase and explains how such differential phosphorylation by different CDKs might translate into a differential regulation of downstream effector pathways.

Published
1997

4. Cyclin-cyclin-dependent kinase regulatory response is linked to substrate recognition.

Author

Cuomo ME, Platt GM, Pearl LH, and Mittnacht S

Subjects
Animals, Cell Line, Cyclin D chemistry, Cyclin D genetics, Cyclin-Dependent Kinase Inhibitor Proteins chemistry, Cyclin-Dependent Kinase Inhibitor Proteins genetics, Cyclin-Dependent Kinases chemistry, Cyclin-Dependent Kinases genetics, Humans, Models, Biological, Models, Molecular, Mutagenesis, Site-Directed, Phosphorylation, Sequence Analysis, Protein, Viral Proteins chemistry, Viral Proteins genetics, Cyclin D metabolism, Cyclin-Dependent Kinase Inhibitor Proteins metabolism, Cyclin-Dependent Kinases metabolism, Evolution, Molecular, Herpesvirus 8, Human enzymology, Viral Proteins metabolism
Abstract

Cyclin/cyclin-dependent kinase (CDK) complexes are critical regulators of cellular proliferation. A complex network of regulatory mechanisms has evolved to control their activity, including activating and inactivating phosphorylation of the catalytic CDK subunit and inhibition through specific regulatory proteins. Primate herpesviruses, including the oncogenic Kaposi sarcoma herpesvirus, encode cyclin D homologues. Viral cyclins have diverged from their cellular progenitor in that they elicit holoenzyme activity independent of activating phosphorylation by the CDK-activating kinase and resistant to inhibition by CDK inhibitors. Using sequence comparison and site-directed mutagenesis, we performed molecular analysis of the cellular cyclin D and the Kaposi sarcoma herpesvirus-cyclin to delineate the molecular mechanisms behind their different behavior. This provides evidence that a surface recognized for its involvement in the docking of CIP/KIP inhibitors is required and sufficient to modulate cyclin-CDK response to a range of regulatory cues, including INK4 sensitivity and CDK-activating kinase dependence. Importantly, amino acids in this region are critically linked to substrate selection, suggesting that a mutational drift in this surface simultaneously affects function and regulation. Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins.

Published
2011
Full Text
View/download PDF

5. Regulation of microfilament organization by Kaposi sarcoma-associated herpes virus-cyclin.CDK6 phosphorylation of caldesmon.

Author

Cuomo ME, Knebel A, Platt G, Morrice N, Cohen P, and Mittnacht S

Subjects
Actins chemistry, Animals, Calmodulin-Binding Proteins metabolism, Catalysis, Chromatography, Affinity, Cloning, Molecular, Cyclin-Dependent Kinase 4 metabolism, Cytoskeleton metabolism, Electrophoresis, Polyacrylamide Gel, HeLa Cells, Humans, Mass Spectrometry, Mice, Microscopy, Fluorescence, NIH 3T3 Cells, Peptides chemistry, Phosphorylation, Protein Binding, Protein Structure, Tertiary, RNA, Small Interfering metabolism, Recombinant Proteins chemistry, Retinoblastoma Protein metabolism, Sepharose chemistry, Serine chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity, Threonine chemistry, Time Factors, Calmodulin-Binding Proteins chemistry, Cyclin-Dependent Kinase 6 metabolism, Gene Expression Regulation, Viral, Herpesvirus 8, Human metabolism
Abstract

Kaposi sarcoma-associated herpes virus (KSHV) encodes a D-like cyclin (K-cyclin) that is thought to contribute to the viral oncogenicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4 and 6, generating enzymes with a substrate selectivity deviant from CDK4 and CDK6 activated by D-type cyclins, suggesting different biochemical and biological functions. Here we report the identification of the actin- and calmodulin-binding protein caldesmon (CALD1) as a novel K-cyclin.CDK substrate, which is not phosphorylated by D.CDK. CALD1 plays a central role in the regulation of microfilament organization, consequently controlling cell shape, adhesion, cytokinesis and motility. K-cyclin.CDK6 specifically phosphorylates four Ser/Thr sites in the human CALD1 carboxyl terminus, abolishing CALD1 binding to its effector protein, actin, and its regulator protein, calmodulin. CALD1 is hyperphosphorylated in cells following K-cyclin expression and in KSHV-transformed lymphoma cells. Moreover, expression of exogenous K-cyclin results in microfilament loss and changes in cell morphology; both effects are reliant on CDK catalysis and can be reversed by the expression of a phosphorylation defective CALD1. Together, these data strongly suggest that K-cyclin expression modulates the activity of caldesmon and through this the microfilament functions in cells. These results establish a novel link between KSHV infection and the regulation of the actin cytoskeleton.

Published
2005
Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results