See also Figure S1, 2 and Table S1. Next, we implemented two complementary and unbiased approaches to identify the proline residue/s on DYRK1 that are hydroxylated by PHD1. VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases and Revefenacin prolyl-hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes. Graphical Abstract In Brief Proline hydroxylation activates DYRK1, which belongs to the CMGC family of protein kinases. Lee et al. identified a highly conserved proline in the kinase domain of CMGC kinases that is hydroxylated by the PHD1 prolyl hydroxylase. Formation of the prolyl Revefenacin hydroxylated intermediate triggers tyrosine autophosphorylation and kinase activation. Introduction Proline hydroxylation is a common but still poorly understood protein modification (Gorres and Raines, 2010). The proline hydroxylation reaction is catalyzed by the 2-oxoglutarate and oxygen dependent dioxygenases PHD1, PHD2 and PHD3 (Semenza, 2001). Rabbit polyclonal to IQCC PHD enzymes hydroxylate HIF1 and HIF2 proteins, thus promoting recognition and destruction of these transcription factors by the Von Hippel Lindau (VHL)-CUL-2 ubiquitin ligase and tumor suppressor (Kaelin and Ratcliffe, 2008). Recently, we uncovered an additional layer of control of HIF in cancer stem cells that is initiated by hydroxylation of the Dual-specificity tyrosine (Y) phosphorylation-regulated kinases 1A (DYRK1A) and 1B (DYRK1B) by PHD1 (Aranda et al., 2011; Lee et al., 2016). PHD1-mediated hydroxylation promoted DYRK1 kinase activity towards a conserved threonine (T27) of ID2, a protein that in its active unphosphorylated form drives the cancer stem cell state and multiple aspects of tumor aggressiveness (Lasorella et al., 2014). When phosphorylated by DYRK1 on T27, ID2 was unable to bind and disrupt the VHL-CUL2 ubiquitin ligase complex, thus restoring VHL function and HIF protein degradation (Lee et al., 2016). However, this work did not identify the DYRK1 proline residue(s) that are directly modified by PHD1 hydroxylation. Consequently, the biochemical events linking PHD1-mediated prolyl hydroxylation of DYRK1 to maturation into a catalytically active kinase remain unknown. DYRK1A and DYRK1B belong to an evolutionary conserved family of protein kinases, the CMGC group, which includes Cyclin dependent kinases (might drive the transformation of nascent kinase polypeptides during or immediately after protein translation into a prone-to-autophosphorylate conformation that would create intermediate forms of the kinases as an essential step in the process of achieving full catalytic activity (Beenstock et al., 2016). The finding that prolyl hydroxylation is sufficient to Revefenacin enhance the catalytic activity of DYRK1 kinases left unanswered the question of when prolyl hydroxylation and tyrosine autophosphorylation occur during DYRK1 maturation and whether they are causally connected. More importantly, our previous findings raised the question as to whether prolyl hydroxylation was a unique property of DYRK1A and DYRK1B or a more general mechanism for activation of protein kinases. Here, we identified a highly conserved proline in the kinase domain of DYRK1 that is hydroxylated by PHD1. Proline hydroxylation precedes and is indispensable for tyrosine autophosphorylation during translation of the DYRK1 polypeptide. We also found that prolyl hydroxylation is necessary for DYRK1 tumor suppression. Finally, we report that prolyl hydroxylation by PHD1 occurs in a conserved domain of CMGC kinases and is an essential early event by which kinases of this large family acquire catalytic activity. Results Identification of the proline residue of DYRK1 hydroxylated by PHD1 To determine whether DYRK1A and DYRK1B are direct substrates of prolyl hydroxylation by PHD1, we first developed an hydroxylation assay to model PHD1-mediated proline hydroxylation of DYRK1. In this system, we incubated baculovirus-expressed GST-DYRK1A or GST-DYRK1B (Figure S1A) in the presence or absence of recombinant MYC-tagged PHD1 in a reaction containing essential cofactors required for the enzymatic activity of PHD prolyl hydroxylases (Selak et al., 2005). As the PHD enzymes require -ketoglutarate as co-substrate (MacKenzie et al., 2007; Schofield and Ratcliffe, 2004), we also tested the effect of -ketoglutarate. Prolyl hydroxylated DYRK1 proteins were recovered by immunoprecipitation with hydroxyl-proline antibody followed by western blot for DYRK1A or DYRK1B. Efficient hydroxylation of GST-DYRK1A and GST-DYRK1B required both -ketoglutarate and recombinant PHD1, thus indicating that prolyl hydroxylation of DYRK1 kinases is mediated by the -ketoglutarate-dependent oxygenase activity of PHD1 (Figure 1A, Figure S1B). PHD1 and -ketoglutarate were also required to induce the enzymatic activity of GST-DYRK1B purified from bacteria as shown by phosphorylation of recombinant FLAG-ID2 using kinase assay (Figure S1C). We confirmed that.