MEKK1-Dependent Activation of the CRL4 Complex Is Important for DNA Damage-Induced Degradation of p21 and DDB2 and Cell Survival.

Cullin-4 ubiquitin ligase (CRL4) complexes are differentially composed and highly dynamic protein assemblies that control many biological processes, including the global genome nucleotide excision repair (GG-NER) pathway. Here, we identified the kinase mitogen-activated protein kinase kinase kinase 1 (MEKK1) as a novel constitutive interactor of a cytosolic CRL4 complex that disassembles after DNA damage due to the caspase-mediated cleavage of MEKK1. The kinase activity of MEKK1 was important to trigger autoubiquitination of the CRL4 complex by K48- and K63-linked ubiquitin chains. MEKK1 knockdown prohibited DNA damage-induced degradation of the CRL4 component DNA-damage binding protein 2 (DDB2) and the CRL4 substrate p21 and also cell recovery and survival. A ubiquitin replacement strategy revealed a contribution of K63-branched ubiquitin chains for DNA damage-induced DDB2/p21 decay, cell cycle regulation, and cell survival. These data might also have implications for cancer, as frequently occurring mutations of MEKK1 might have an impact on genome stability and the therapeutic efficacy of CRL4-dependent immunomodulatory drugs such as thalidomide derivatives.

The WD40-repeat protein Han11 functions as a scaffold protein to control HIPK2 and MEKK1 kinase functions.

Protein kinases are organized in hierarchical networks that are assembled and regulated by scaffold proteins. Here, we identify the evolutionary conserved WD40-repeat protein Han11 as an interactor of the kinase homeodomain-interacting protein kinase 2 (HIPK2). In vitro experiments showed the direct binding of Han11 to HIPK2, but also to the kinases DYRK1a, DYRK1b and mitogen-activated protein kinase kinase kinase 1 (MEKK1). Han11 was required to allow coupling of MEKK1 to DYRK1 and HIPK2. Knockdown experiments in Caenorhabditis elegans showed the relevance of the Han11 orthologs Swan-1 and Swan-2 for the osmotic stress response. Downregulation of Han11 in human cells lowered the threshold and amplitude of HIPK2- and MEKK1-triggered signalling events and changed the kinetics of kinase induction. Han11 knockdown changed the amplitude and time dependence of HIPK2-driven transcription in response to DNA damage and also interfered with MEKK1-triggered gene expression and stress signalling. Impaired signal transmission also occurred upon interference with stoichiometrically assembled signalling complexes by Han11 overexpression. Collectively, these experiments identify Han11 as a novel scaffold protein regulating kinase signalling by HIPK2 and MEKK1.