Upon initial sequencing and analysis of Rhes, we found the protein contains PtdIns (3,4,5)and a P3
) binding site (www.scansite.mit.edu
). Growth factors activate PI3 kinase generating PIP3
. Because it is well established that AKT is the primary target of PI3K [4
], we first investigated whether Rhes has any effect on PI3K/AKT pathway. We overexpressed either Myc-Rhes or Myc plasmid, for control, in HEK293T cells and stimulated cells with serum. We found that Rhes augments serum-mediated activation of AKT, as determined by phosphorylation of AKT (). In order to examine the specificity of the apparent growth factor effect, we treated HEK293T cells overexpressed with Myc-Rhes or Myc control with individual growth factors. We observed that all growth factors tested: IGF-1, EGF, PDGF and FGF, enhanced AKT activation in the presence of Rhes overexpression more so than in the Myc control condition. This suggests that the effect of Rhes is not growth factor specific (). We also constructed a stable Rhes-inducible cell line to eliminate the heterogeneity of transient transfection. Inducible T-REx293 cells were treated with tetracycline for 6h to induce Rhes expression and incubated with IGF-1 for 10 min (). We observed that acutely induced Rhes enhanced phosphorylation of AKT. To examine the physiologic relevance of Rhes function, we compared the influence of Rhes overexpression on EGF-induced AKT activation in differentiated rat phaeochromocytoma (PC12) cells. We found that overexpression of Rhes enhanced phosphorylation of Akt (T308) in differentiated PC 12 cells ().
Rhes-mediated AKT activation by growth factors
To investigate the potential mechanism by which Rhes activates the AKT pathway, we first examined whether Rhes interacts with AKT. We overexpressed GST-Rhes into HEK293T cells and performed GST-pull down assay. We found that endogenous AKT physically interacts with GST-Rhes (Supplementary Fig. 1A
). Rhes contains a PIP3
binding site which can interact with the Pleckstrin homology (PH) domain. We then questioned whether the PH-domain of AKT is involved in the interaction between Rhes and AKT. We overexpressed GST-Rhes and either a fragment of AKT PH domain, or a mutant fragment of AKT PH domain (R25C) which does not bind to PIP3
nor translocate to the membrane upon serum stimulation. We found that Rhes only interacts with wild type AKT PH domain (Supplementary Fig. 1B
To further understand how Rhes participates in PI3K/AKT pathway, we tested whether Rhes modulates PI3K activity to influence AKT signaling. An essential process for PI3K activation is its recruitment to the plasma membrane, whereby PI3K can access its substrate such as PIP2
]. To examine this process, we measured levels of PIP3
in a competitive ELISA but no change was detected in PIP3
levels in the presence of overexpressed Rhes. This lack of change suggests that Rhes does not affect PI3K activity. We next examined whether Rhes can physically interact with PI3K. At the basal state, p85/PI3K is required to stabilize the catalytic p110 subunit of PI3K [24
]. By overexpressing GST-Rhes and Flag-p85/PI3K plasmids, we found that p85/PI3K interacts with Rhes in HEK293T cells in a GST pull down assay ().
Rhes interacts with p85 regulatory subunit of PI3K
In order to identify where Rhes binds p85/PI3K, we generated fragments of Rhes fused with GST and probed against p85/PI3K in a GST pull-down assay. We found that C-terminal tail (210–266), the unique region for RASD1 family, contains the PIP3 binding domain and is responsible for the interaction between Rhes and p85/PI3K (). Moreover, we found that the interaction between p85/PI3K and Rhes gradually increases upon IGF-1 treatment, while cells overexpressing the Rhes mutant, without the binding C-terminal tail, does not interact with p85 at all (). Interestingly, we found that IGF-1-mediated association between p85/PI3K and Rhes is solely mediated by C-terminal end (210–266) ().
Upon growth factor treatment, AKT is translocated to the membrane from cytosol and is phosphorylated at Thr308 and Ser473[4
]. We then examined whether Rhes has an effect on AKT translocation to membrane. To elucidate this type of interaction, HEK293T cells overexpressed with Myc-Rhes or Myc control were treated with 10 nM insulin for 15 min, followed by subcellular fractionation. By examining each of the fractions through western blotting, we found that AKT translocation to membrane is enhanced in cells overexpressed with Rhes (). To further examine the effect Rhes has on AKT translocalization, we transfected HEK293 cells containing AKT-PH-EGFP with either Myc-Rhes or a Myc control plasmids, and treated with low dose of insulin (10 nM for 15 min). We observed that AKT is completely translocated to the membrane in cells overexpressing Rhes while cells expressing the Myc control plasmid showed only partial translocation of AKT to membrane after insulin treatment ().
Growth factor stimulation induces Rhes-mediated AKT activation and subcellular localization