The multitude of mechanisms regulating the activity of protein kinases includes phosphorylation of amino acids contained in the activation loop. Here we show that the serine/threonine kinase HIPK2 (homeodomain-interacting protein kinase 2) is heavily modified by autophosphorylation, which occurs by cis-autophosphorylation at the activation loop and by trans-autophosphorylation at other phosphorylation sites. Cis-autophosphorylation of HIPK2 at Y354 and S357 in the activation loop is essential for its kinase function and the binding to substrates and the interaction partner Pin1. HIPK2 activation loop phosphorylation is also required for its biological activity as a regulator of gene expression and cell proliferation. Phosphorylation of HIPK2 at Y354 alone is not sufficient for full HIPK2 activity, which is in marked contrast to some dual-specificity tyrosine-phosphorylated and regulated kinases where tyrosine phosphorylation is absolutely essential. This study shows that differential phosphorylation of HIPK2 provides a mechanism for controlling and specifying the signal output from this kinase.

RNA viruses have rapidly evolving genomes which often allow cross-species transmission and frequently generate new virus variants with altered pathogenic properties. Therefore infections by RNA viruses are a major threat to human health. The infected host cell detects trace amounts of viral RNA and the last years have revealed common principles in the biochemical mechanisms leading to signal amplification that is required for mounting of a powerful antiviral response. Components of the RNA sensing and signaling machinery such as RIG-I-like proteins, MAVS and the inflammasome inducibly form large oligomers or even fibers that exhibit hallmarks of prions. Following a nucleation event triggered by detection of viral RNA, these energetically favorable and irreversible polymerization events trigger signaling cascades leading to the induction of antiviral and inflammatory responses, mediated by interferon and NF-κB pathways. Viruses have evolved sophisticated strategies to manipulate these host cell signaling pathways in order to ensure their replication. We will discuss at the examples of influenza and HTLV-1 viruses how a fascinating diversity of biochemical mechanisms is employed by viral proteins to control the NF-κB pathway at all levels.

The serine/threonine kinase homeodomain-interacting protein kinase (HIPK2) is a tumor suppressor and functions as an evolutionary conserved regulator of signaling and gene expression. This kinase regulates a surprisingly vast array of biological processes that range from the DNA damage response and apoptosis to hypoxia signaling and cell proliferation. Recent studies show the tight control of HIPK2 by hierarchically occurring posttranslational modifications such as phosphorylation, small ubiquitin-like modifier modification, acetylation, and ubiquitination. The physiological function of HIPK2 as a regulator of cell proliferation and survival has a downside: proliferative diseases. Dysregulation of HIPK2 can result in increased proliferation of cell populations as it occurs in cancer or fibrosis. We discuss various models that could explain how inappropriate expression, modification, or localization of HIPK2 can be a driver for these proliferative diseases.

The multi-subunit CCR4 (carbon catabolite repressor 4)-NOT (Negative on TATA) complex serves as a central coordinator of all different steps of eukaryotic gene expression. Here we performed a systematic and comparative analysis of cells where the CCR4-NOT subunits CNOT1, CNOT2 or CNOT3 were individually downregulated using doxycycline-inducible shRNAs. Microarray experiments showed that downregulation of either CNOT subunit resulted in elevated expression of major histocompatibility complex class II (MHC II) genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of either CNOT subunit was seen in a variety of cell systems and also in naïve macrophages from CNOT3 conditional knock-out mice. CNOT2-mediated repression of MHC II genes occurred also in the absence of the master regulator class II transactivator (CIITA) and did not cause detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs whereas tethering of CNOT2 to a regulatory region governing MHC II expression resulted in diminished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors restricting class II expression.

The non-canonical IKK kinase TBK1 serves as an important signal transmitter of the antiviral interferon response, but is also involved in the regulation of further processes such as autophagy. The activity of TBK1 is regulated by posttranslational modifications comprising phosphorylation and ubiquitination. This study identifies SUMOylation as a novel posttranslational TBK1 modification. TBK1 kinase activity is required to allow the attachment of SUMO1 or SUMO2/3 proteins. Since TBK1 does not bind to the E2 enzyme Ubc9, this modification most likely proceeds via trans-SUMOylation. Mass spectrometry allowed identifying K694 as the SUMO acceptor site, a residue located in the C-terminal coiled-coil domain which is exclusively responsible for the association with the adaptor proteins NAP1, Sintbad and TANK. SUMO modification at K694 contributes to the antiviral function of TBK1 and accordingly the viral protein Gam1 antagonizes this posttranslational modification.