Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis

Prasanna Parasuraman,Peter Mulligan,James A Walker,Bihua Li,Myriam Boukhali,Wilhelm Haas,Andre Bernards

doi:10.1074/jbc.m116.769216

Prasanna Parasuraman, Peter Mulligan + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m116.769216

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Abstract

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Highlights

Members of the Rho GTPase family act as binary switches to control many cell biological processes
Given the importance of RhoA-C, Rac1–3, Cdc42, and other Rho family members in cell biology, it may not be surprising that they are subject to intricate regulation
This is perhaps best illustrated by the fact that the human genome includes 68 genes encoding RhoGAP-like proteins, and an even larger number or genes predicting RhoGEFs [2, 33, 34]

Summary

Results

Proteomic Identification of p190A-associated Proteins—In HeLa cell extracts fractionated by differential centrifugation, p190A predominantly resides in the detergent-soluble membrane fraction, with some protein found in the nuclear pellet and cytosol (Fig. 1A). Confirmation of the p190A/eIF3 Association—To rule out an obvious artifact, we tested whether the rabbit polyclonal antibody used to affinity purify p190A complexes cross-reacts with one or more translation factors To rule this out, we used a mouse monoclonal antibody to precipitate p190A from HeLa cells, and analyzed the presence of eIF3A, eIF3B, eIF3C, eIF3D, and eIF3H by immunoblotting. This first FF domain includes Tyr308, whose PDGF-stimulated phosphorylation was previously found to disrupt an interaction with transcription factor TFII-I [16] For this reason, and because growth factor stimulation appeared to cause an increase in the amount of several proteins associated with p190A (Table 1 and supplemental Table S1), we analyzed whether ␭-phosphatase treatment of HeLa extracts prior to immunoprecipitation affected the p190A-eIF3A association. When an anti-HA antibody was used to precipitate these proteins from HeLa cells, the wild-type and S296D mutant proteins co-precipitated similar amounts of GTPase 1-268

10 AA Repeat

Discussion

Experimental Procedures