SPAK (STE20/SPS1-related Pro/Ala-rich kinase) and OSR1 (oxidative stress responsive 1) are members of the GCK family serine/threonine kinases. Overexpression and in vitro studies demonstrate that SPAK is able to activate p38 MAP kinase indicating a possible role for SPAK in the stress response. Yeast two-hybrid screening revealed that SPAK and OSR1 bind to Na-K-2Cl cotransporters NKCC1 and NKCC2 and K-Cl cotransporter KCC3. WNK1 and WNK4 phosphorylate SPAK at Thr243/247 and Ser380. Similarly, WNK1 and WNK4 phosphorylate OSR1 at Thr185 and Ser315. Phosphorylation at these sites stimulates SPAK and OSR1 activity, leading to NKCC1 phosphorylation and enhanced NKCC1 activity. SPAK is also phosphorylated at Ser311 by PKCtheta in response to T cell activation. Substitution of Ser311 with Ala or specific siRNA knock-down of SPAK dramatically reduces TCR/CD28-induced AP-1 activation, suggesting SPAK is involved in T cell signaling as well.
