Abstract
Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.
Highlights
The Rho family of GTPases, part of the Ras superfamily, contains multiple subgroups, including the Ras-related C3 botulinum toxin substrate (Rac), cell division control protein 42 homolog (CDC42), and Rho-like GTPases [1]
In MCF7 breast cancer cells, we found that insulin caused a PREX1 mobility shift (Fig. 1A)
Treatment with -phosphatase ablated the receptor tyrosine kinases (RTKs)-induced electrophoretic mobility shift, demonstrating that these changes to PREX1 were the result of phosphorylation (Fig. 1B). -Phosphatase treatment compressed band 2, which is seen in the starved state, indicating that steady-state phosphorylation is present on PREX1
Summary
PREX1 Is Phosphorylated through a PI3K-dependent Mechanism Downstream of Receptor Tyrosine Kinase Activation— PREX1 is phosphorylated downstream of neuregulin and IGF1 [18, 19], and multiple studies have demonstrated that phosphorylation causes an electrophoretic mobility shift in PREX1 [18, 30, 31]. Treatment of MCF7 cells with the PAK inhibitor PF-3758309 or co-expression of the dominant negative PAK1 K299R mutant reduced enrichment of the upper PREX1 band (band 3) that was induced by isoproterenol or PGE2, suggesting that these agonists do cause PAK-dependent phosphorylation events (Fig. 4, D and E). MCF7 cells contain an activating mutation on the catalytic subunit of PI3K␣, and these data indicate that the basal levels of PIP3 that likely result from this mutation are important for the stimulation of PAK that occurs downstream of GPCRs and PKA activation Taken together, these data demonstrate that PAKs have a role in the phosphorylation of PREX1 downstream of isoproterenol and PGE2; the mechanism and consequences of phosphorylation, perhaps to the differential role of PKA, appear to be different from the phosphorylation of PREX1 downstream of RTKs
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