Adaptation to Endoplasmic Reticulum Stress Requires Trans-phosphorylation within the Activation Loop of Protein Kinases Kin1 and Kin2, Orthologs of Human Microtubule Affinity-regulating Kinase.

Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed the "ER stress", activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog Kin2 in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity-regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an auto-inhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for the UPR function. We also show that the functional mini Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that trans-phosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for the full kinase activity. Collectively, our data suggest that, during the ER stress, the Kin kinase domain is released from its auto-inhibitory KA1 domain and is activated by trans-phosphorylation.