Abstract
Protein kinases are essential molecules in life and their crucial function requires tight regulation. Many kinases are regulated via phosphorylation within their activation loop. This loop is embedded in the activation segment, which additionally contains the Mg2+ binding loop and a P + 1 loop that is important in substrate binding. In this report, we identify Abl-mediated phosphorylation of a highly conserved Tyr residue in the P + 1 loop of protein kinase D2 (PKD2) during oxidative stress. Remarkably, we observed that the three human PKD isoforms display very different degrees of P + 1 loop Tyr phosphorylation and we identify one of the molecular determinants for this divergence. This is paralleled by a different activation mechanism of PKD1 and PKD2 during oxidative stress. Tyr phosphorylation in the P + 1 loop of PKD2 increases turnover for Syntide-2, while substrate specificity and the role of PKD2 in NF-κB signaling remain unaffected. Importantly, Tyr to Phe substitution renders the kinase inactive, jeopardizing its use as a non-phosphorylatable mutant. Since large-scale proteomics studies identified P + 1 loop Tyr phosphorylation in more than 70 Ser/Thr kinases in multiple conditions, our results do not only demonstrate differential regulation/function of PKD isoforms under oxidative stress, but also have implications for kinase regulation in general.
Highlights
Protein kinases are essential as receivers, transmitters and executors of a wide variety of cellular stimuli
Because the protein kinase D (PKD) family of kinases is regulated by tyrosine phosphorylation during oxidative stress, we investigated whether Tyr-717 could be phosphorylated under these conditions
Tyr-717 phosphorylation could be detected with sub-millimolar doses of H2O2 (Fig. 1e), but no phosphorylation was seen during classical activation of protein kinase D2 (PKD2) with GPCR agonists or phorbol-12,13-dibutyrate (PDB) (Fig. 1f)
Summary
PKD2 is phosphorylated at Tyr-717 in the activation segment under oxidative stress conditions. This discrepancy was only observed in oxidative stress stimulated enzymes, since in PDB stimulated conditions both isoforms share similar kinetics (Fig. 6b and Table 1) To examine whether this difference in activity could be due to Tyr-717 phosphorylation in PKD2, we made use of the aforementioned PKD2 LNQ.RNK exchange mutant, which shows a strong reduction in Tyr-717 phosphorylation compared to WT PKD2, without aberrations in activation loop Ser-706/710 phosphorylation (Fig. 6c). Kinetic analysis of purified PKD2 from Abl overexpressing cells treated with or without STI-571 revealed an increase in kcat values for PKD2 phosphorylated at Tyr-717 by about 1.5 fold compared to a non-Tyr phosphorylated species (Fig. 7b and Table 2) This increase is independent of the activation loop Ser phosphorylation state, which is at similar (basal) levels in both conditions (Fig. 7c). Consistent with this idea, increasing PKD2 activity in oxidative stress does not affect PKD2 signaling to NF-κB, as shown with a S706/710E mutant (Fig. 8a)
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