Phosphorylation of RACK1 in plants

Abstract

Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. These findings promote a new regulatory system in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.

Keywords:

• Arabidopsis
• kinase
• phosphorylation
• protein degradation
• RACK1
• scaffold protein
• WNK8

Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades in mammalian cells.^1–7 RACK1 protein contains a 7 tryptophan–aspartic acid repeat domain (WD40) similar to that in the heterotrimeric GTP-binding protein β subunit. The seven blade propeller structure of RACK1 allows it to function as a signaling hub.⁴

The protein sequences and the structure of RACK1 are conserved in plants.^8,9 Similar to mammalian RACK1, plant RACK1 interacts with a large group of proteins ^10,11 and is involved in diverse biological processes, ranging from seed germination, leaf and root development to flowering,^8,12 and is also involved in responses to plant hormones, ^8,13 and biotic and abiotic stresses.^13–15 At the molecular level, RACK1 protein is associated with ribosomes ^16-18 and is involved in the regulation of protein translation ¹⁸ and miRNA abundance.¹⁹

It is less clear how RACK1 is able to participate in so many different biological processes. Studies using mammalian cells suggested a few possible mechanisms including translocation of RACK1 protein from one cellular compartment to another, formation of protein complex with other scaffolding proteins and contribution to transcription and translation.^4,6 These abilities of RACK1 lead to its temporal and spatial regulation of diverse signal transduction events. Emerging evidence suggest that the transcriptional and translational modification of RACK1 itself also play an important role in regulating its function. For example, the transcription of RACK1 is suppressed by the plant stress hormone abscisic acid (ABA).¹³ It was suggested that such transcriptional regulation likely contributes to RACK1's negative regulation of ABA responses in the ABA-mediated inhibition of seed germination, early seedling development, and ABA-induced gene expression.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.^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. Therefore, mammalian RACK1 is a substrate of tyrosine kinase whereas plant RACK1 is a substrate of serine/threonine kinase. This finding may not be surprising since for plant proteins, most phosphorylation occurs on Ser and Thr and to a less extent on Tyr.²³

It should be noted that the Tyr phosphorylation site of mammalian RACK1 is also required for its binding to its Tyr kinase. 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. The study by Urano et al. (2015) ²⁰ also does not rule out the possibility that other phosphorylation sites may exist in RACK1 protein. Amino acid sequence alignment indicated that Tyr phosphorylation sites identified in mammalian RACK1 (Tyr52, Tyr228 and Tyr246) are conserved in plant RACK1 proteins.²⁰ Whether RACK1 is phosphorylated by a yet unidentified Tyr kinase in plants remains an open question.

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. 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). Specifically, nonphosphorylated RACK1 positively regulate flowering and negatively regulate the response to high concentration of glucose. Upon phosphorylation by WNK8, RACK1 protein becomes unstable and undergoes protein degradation. Presumably, RACK1 is degraded through the 26S proteasome but this has not been experimentally examined. The phosphatase that specifically de-phosphorylates phosphorylated RACK1 by WNK8, however, remains to be identified. It is speculated that such a phosphatase counteracts the phosphorylation of RACK1 by WNK8, providing a positive feedback loop. The assumption of presence of other forms of posttranslational modification events for RACK1 is consistent with the fact that no difference in RACK1 protein abundance was detected between wnk8 mutant and the wild type.²⁰

Figure 1. Phosphorylation of RACK1 by WNK8. WNK8 interacts with and phosphorylates RACK1 at Ser122 and Thr162. Phosphorylation of RACK1 promotes protein degradation. An unidentified phosphatase counteracts RACK1 phosphorylation by WNK8. Nonphosphorylated RACK1 protein acts downstream of WNK8 to regulate flowering and response to high concentration of glucose.

The finding by Urano et al. (2015) ²⁰ provides new insights into the molecular mechanism underlying the regulatory actions of RACK1. This also opens up a few more questions: (1) How RACK1 phosphorylation by WNK8 is regulated? (2) What is the phosphatase that counteracts RACK1 phosphorylation by WNK8? (3) Is RACK1 protein subject to Tyr phosphorylation by an unidentified Tyr kinase? (4) Is RACK1 protein subject to other forms of posttranslational modifications? Addressing these questions will drastically enhance our understanding of the molecular mechanism of action of RACK1 in plants.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science Program, United States Department of Energy, Office of Science, Biological and Environmental Research. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the United States Department of Energy under contract DE-AC05–00OR22725.