Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

Abstract

Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. Here, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1A S122A/T162A, but not the phosphomimetic form, RACK1A S122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1A S122D/T162E accumulated at similar levels as those of RACK1 S122A/T162A . However, although the steady-state level of the RACK1A S122A/T162A protein was similar to wild-type RACK1A protein, the RACK1A S122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. Taken together, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.

Phosphorylation of RACK1 in plants

“…While the post-translational modification events have previously reported for mammalian RACK1 protein, 21,22 its impact on protein stability has not. The finding by Urano et al (2015) 20 promotes a new regulatory system in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation (Fig. 1).…”

“…However, it remains unknown whether Ser122 and Thr162 are also required for RACK1 binding to WNK8. The study by Urano An important finding of the study by Urano et al (2015) 20 is that phosphorylation of RACK1 affects its protein stability. While the post-translational modification events have previously reported for mammalian RACK1 protein, 21,22 its impact on protein stability has not.…”

“…13 More recently, it was demonstrated that RACK1 is also regulated at the post-translational level. 20 It was found that RACK1 protein is subject to phosphorylation and that phosphorylation of RACK1 renders it unstable. 20 Phosphorylation of RACK1 has been previously reported in mammals.…”

“…20 It was found that RACK1 protein is subject to phosphorylation and that phosphorylation of RACK1 renders it unstable. 20 Phosphorylation of RACK1 has been previously reported in mammals. [21][22] However, a fundamental difference is that mammalian RACK1 is phosphorylated at tyrosine (Tyr) sites whereas plant RACK1 is phosphorylated at serine (Ser) and threonine (Thr) sites.…”

“…Specifically, Tyr228 and Tyr246 are required for RACK1 binding to Src kinase 21 and Tyr52 of RACK1 is required for its binding to c-Abl kinase. 22 Urano et al (2015) 20 showed that Ser122 and Thr162 (Thr162 of RACK1A, Thr161 of RACK1B and RACK1C) of Arabidopsis RACK1 are phosphorylated by an atypical Ser/Thr kinase, WITH NO LYSINE 8 (WNK8). However, it remains unknown whether Ser122 and Thr162 are also required for RACK1 binding to WNK8.…”