The Protein Interaction Landscape of the Human CMGC Kinase Group

Peer reviewe

Kinase Interaction Network Expands Functional and Disease Roles of Human Kinases

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.Peer reviewe

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca(2+) release and store-operated Ca(2+) entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1microM) and the Src inhibitor PP2 (10microM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca(2+) transients were reduced by imatinib and/or PP2. Ca(2+) transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca(2+) transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCalpha catalytic activity and PKCalpha co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca(2+) influx was reduced by complexing extracellular Ca(2+) with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca(2+) transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells

The MST1 and hMOB1 Tumor Suppressors Control Human Centrosome Duplication by Regulating NDR Kinase Phosphorylation

BACKGROUND: Human MST/hSAV/LATS/hMOB tumor suppressor cascades are regulators of cell death and proliferation; however, little is known about other functions of MST/hMOB signaling. Mob1p, one of two MOB proteins in yeast, appears to play a role in spindle pole body duplication (the equivalent of mammalian centrosome duplication). We therefore investigated the role of human MOB proteins in centrosome duplication. We also addressed the regulation of human centrosome duplication by mammalian serine/threonine Ste20-like (MST) kinases, considering that MOB proteins can function together with Ste20-like kinases in eukaryotes. RESULTS: By studying the six human MOB proteins and five MST kinases, we found that MST1/hMOB1 signaling controls centrosome duplication. Overexpression of hMOB1 caused centrosome overduplication, whereas RNAi depletion of hMOB1 or MST1 impaired centriole duplication. Significantly, we delineated an hMOB1/MST1/NDR1 signaling pathway regulating centrosome duplication. More specifically, analysis of shRNA-resistant hMOB1 and NDR1 mutants revealed that a functional NDR/hMOB1 complex is critical for MST1 to phosphorylate NDR on the hydrophobic motif that in turn is required for human centrosome duplication. Furthermore, shRNA-resistant MST1 variants revealed that MST1 kinase activity is crucial for centrosome duplication whereas MST1 binding to the hSAV and RASSF1A tumor suppressor proteins is dispensable. Finally, by studying the PLK4/HsSAS-6/CP110 centriole assembly machinery, we also observed that normal daughter centriole formation depends on intact MST1/hMOB1/NDR signaling, although HsSAS-6 centriolar localization is not affected. CONCLUSIONS: Our observations propose a novel pathway in control of human centriole duplication after recruitment of HsSAS-6 to centrioles

BRAF inhibition sensitizes melanoma cells to α-amanitin via decreased RNA polymerase II assembly

Despite the great success of small molecule inhibitors in the treatment of patients with BRAF mutated melanoma, the response to these drugs remains transient and patients eventually relapse within a few months, highlighting the need to develop novel combination therapies based on the understanding of the molecular changes induced by BRAF inhibitors. The acute inhibition of oncogenic signaling can rewire entire cellular signaling pathways and thereby create novel cancer cell vulnerabilities. Here, we demonstrate that inhibition of BRAF oncogenic signaling in melanoma cell lines leads to destabilization of the large subunit of RNA polymerase II POLR2A (polymerase RNA II DNA-directed polypeptide A), thereby preventing its binding to the unconventional prefoldin RPB5 interactor (URI1) chaperone complex and the successful assembly of RNA polymerase II holoenzymes. Furthermore, in melanoma cell lines treated with mitogen-activated protein kinase (MAPK) inhibitors, α-amanitin, a specific and irreversible inhibitor of RNA polymerase II, induced massive apoptosis. Pre-treatment of melanoma cell lines with MAPK inhibitors significantly reduced IC50 values to α-amanitin, creating a state of collateral vulnerability similar to POLR2A hemizygous deletions. Thus, the development of melanoma specific α-amanitin antibody-drug conjugates could represent an interesting therapeutic approach for combination therapies with BRAF inhibitors

BRAF inhibition sensitizes melanoma cells to α-amanitin via decreased RNA polymerase II assembly

Despite the great success of small molecule inhibitors in the treatment of patients with BRAFV600E mutated melanoma, the response to these drugs remains transient and patients eventually relapse within a few months, highlighting the need to develop novel combination therapies based on the understanding of the molecular changes induced by BRAFV600E inhibitors. The acute inhibition of oncogenic signaling can rewire entire cellular signaling pathways and thereby create novel cancer cell vulnerabilities. Here, we demonstrate that inhibition of BRAFV600E oncogenic signaling in melanoma cell lines leads to destabilization of the large subunit of RNA polymerase II POLR2A (polymerase RNA II DNA-directed polypeptide A), thereby preventing its binding to the unconventional prefoldin RPB5 interactor (URI1) chaperone complex and the successful assembly of RNA polymerase II holoenzymes. Furthermore, in melanoma cell lines treated with mitogen-activated protein kinase (MAPK) inhibitors, α-amanitin, a specific and irreversible inhibitor of RNA polymerase II, induced massive apoptosis. Pre-treatment of melanoma cell lines with MAPK inhibitors significantly reduced IC50 values to α-amanitin, creating a state of collateral vulnerability similar to POLR2A hemizygous deletions. Thus, the development of melanoma specific α-amanitin antibody-drug conjugates could represent an interesting therapeutic approach for combination therapies with BRAFV600E inhibitors.ISSN:2045-232

Autoregulatory control of the p53 response by caspase-mediated processing of HIPK2

The serine/threonine kinase HIPK2 phosphorylates the p53 protein at Ser 46, thus promoting p53-dependent gene expression and subsequent apoptosis. Here, we show that DNA damaging chemotherapeutic drugs cause degradation of endogenous HIPK2 dependent on the presence of a functional p53 protein. Early induced p53 allows caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977. The resulting C-terminally truncated HIPK2 forms show an enhanced induction of the p53 response and cell death, thus allowing the rapid amplification of the p53-dependent apoptotic program during the initiation phase of apoptosis by a regulatory feed-forward loop. The active HIPK2 fragments are further degraded during the execution and termination phase of apoptosis, thus ensuring the occurrence of HIPK2 signaling only during the early phases of apoptosis induction