Interaction of Farnesylated PRL-2, a Protein-tyrosine Phosphatase, with the β-Subunit of Geranylgeranyltransferase II

Xiaoning Si,Qi Zeng,Chee Hoe Ng,Wanjin Hong,Catherine J Pallen

doi:10.1074/jbc.m010400200

Xiaoning Si, Qi Zeng + Show 3 more

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https://doi.org/10.1074/jbc.m010400200

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Abstract

Protein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the beta-subunit of Rab geranylgeranyltransferase II (betaGGT II). The specific interaction of betaGGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with betaGGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for betaGGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for betaGGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the alpha-subunit (alpha) of GGT II, betaGGT II, and PRL-2 resulted in alpha/betaGGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous alpha/betaGGT II activity in HeLa cells. Together, these results indicate that the binding of alphaGGT II and PRL-2 to betaGGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity.

Highlights

Protein prenylation is a post-translational modification with an important role in targeting proteins to membranes and in protein-protein interactions
A human HeLa cell cDNA library fused to the GAL4 activating domain under the control of the constitutive alcohol dehydrogenase 1 promoter was transfected into Y190 cells that expressed DNA-binding domain-Protein of regenerating liver (PRL)-2
The association of PRL-2 with ␤GGT II depends on PRL-2 prenylation, as a potent farnesyltransferase inhibitor, FT inhibitors (FTI)-277 [33], can abolish the association (Fig. 4B)

Summary

Introduction

Protein prenylation is a post-translational modification with an important role in targeting proteins to membranes and in protein-protein interactions (reviewed in Refs. 1 and 2). These results indicate that the binding of ␣GGT II and PRL-2 to ␤GGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity. To determine whether the linked properties of PRL-2 prenylation status and subcellular localization affect its interaction with GGT II, co-immunoprecipitation experiments were carried out from lysates of HeLa cells expressing FLAG-tagged ␤GGT II and Myc-tagged PRL-2 or PRL-2(cd) mutant.

Results

Conclusion