We discovered that RGS13 is phosphorylated by PKA at Thr41 and that activation of the cAMP–PKA pathway promoted RGS13 protein expression in cells dramatically. PKA activation may reduce RGS13 protein turnover through a mechanism dependent on the phosphorylation of RGS13 at Thr41. Mutation of this residue not only decreased PKA-mediated phosphorylation in vitro, but also significantly reduced the steady-state expression of RGS13 protein. Phosphorylation appeared to protect RGS13 from degradation downstream of ubiquitylation as RGS13 ubiquitylation was not affected by cAMP. These studies provide the first insight into mechanisms of post-translational modulation of RGS13 abundance.
PKA has been shown to phosphorylate other RGS proteins such as RGS10, RGS9-1 and RGS14 (Balasubramanian et al., 2001
; Hollinger et al., 2003
). RGS10 phosphorylation by PKA induced its nuclear translocation and crippled its ability to inhibit inwardly rectifying K+
(GIRK) channels in Xenopus laevis
oocytes by sequestration from membrane channels (Burgon et al., 2001
; Bender et al., 2008
). In contrast to RGS10, RGS13 phosphorylation by PKA seems to promote RGS13 function through stabilization of its expression. Whereas mutation of the PKA target residue in RGS13 (Thr41) did not affect its ability to interact with other signaling proteins [pCREB (Figure ) and Gα (data not shown)], the RGS13(T41A) had reduced steady-state expression (Figure ), increased turnover in the presence of cAMP (Figure ) and decreased capacity to inhibit GCPR signaling (Figure ).
Although RGS13 lacks a clear consensus PKA phosphorylation site, we identified Thr41 as a PKA phosphorylation site in RGS13 by mass spectrometry (MS). Mutation of this residue significantly decreased, but did not abolish, RGS13 phosphorylation by PKA, suggesting that additional PKA phosphorylation sites may exist in RGS13 and could contribute to the inhibition of RGS13 degradation by PKA activation. On the other hand, protection of RGS13 degradation by cAMP was completely reversed both by mutation of Thr41 and by expression of PKI, a peptide that specifically inhibits PKA kinase activity in vivo
but not the activity of closely related kinases (Gudi et al., 1996
). These results indicated that cAMP inhibited RGS13 degradation solely by activation of PKA but not other cAMP-activated mediators such as Epac (Gloerich and Bos, 2010
) and that such inhibition most likely occurs through phosphorylation of Thr41 in RGS13 by PKA.
Although Ser/Thr phosphorylation may affect protein ubiquitylation and proteasomal degradation of some proteins (Hino et al., 2005
), RGS13 phosphorylation by PKA did not inhibit RGS13 ubiquitylation. Ubiquitylated proteins may be relatively stable unless they also contain an unstructured region, which is recognized by the 19S regulatory component of the proteasome complex (Schrader et al., 2009
). Thus, phosphorylation of RGS13 at Thr41 by PKA could render the N-terminus more structured overall, impairing recognition and/or handling of ubiquitylated RGS13 by the proteasomal machinery. We hypothesized that this mechanism could underlie the stabilization of the related protein RGS16 by phosphorylation of a conserved Tyr residue at its C-terminus, which is in close proximity to the N-terminus (Derrien et al., 2003
). PKA-induced phosphorylation could also protect RGS13 from proteasomal targeting indirectly by affecting protein–protein interactions. PKA activation induces RGS13 translocation to the nucleus and binding to pCREB. We found previously that extinction of CREB expression by RNAi also reduced RGS13 nuclear localization in cells activated by PKA (Xie et al., 2008
). Therefore, the amount of free cytosolic RGS13 available for proteasome-mediated degradation could be reduced in the presence of PKA.
Owing to their powerful ability to modulate numerous cellular signaling pathways, RGS protein concentrations are often regulated by induction or repression of their transcription by many environmental stimuli. As our studies with RGS13 demonstrate, RGS protein degradation, as is true for many signaling molecules, is also rapidly and precisely coordinated by post-transcriptional and post-translational events. This study lays the foundation for further discovery of unique molecular components that modulate RGS13 longevity and possibly other mechanisms that affect its expression and/or function.