Abstract
PRL-1, -2, and -3 represent a novel class of protein-tyrosine phosphatase with a C-terminal prenylation motif. Although PRL-1 has been suggested to be associated with the nucleus, the presence of three highly homologous members and the existence of a prenylation motif call for a more detailed examination of their subcellular localization. In the present study, we first demonstrate that mouse PRL-1, -2, and -3 are indeed prenylated. Examination of N-terminal epitope-tagged PRL-1, -2, and -3 expressed in transiently transfected cells suggests that PRL-1, -2, and -3 are present on the plasma membrane and intracellular punctate structures. Stable Chinese hamster ovary cells expressing PRL-1 and -3 in an inducible manner were established. When cells were treated with brefeldin A, PRL-1 and -3 accumulated in a collapsed compact structure around the microtubule-organizing center. Furthermore, PRL-1 and -3 redistributed into swollen vacuole-like structures when cells were treated with wortmannin. These characteristics of PRL-1 and -3 are typical for endosomal proteins. Electron microscope immunogold labeling reveals that PRL-1 and -3 are indeed associated with the plasma membrane and the early endosomal compartment. Expression of PRL-3 is detected in the epithelial cells of the small intestine, where PRL-3 is present in punctate structures in the cytoplasm. When cells are treated with FTI-277, a selective farnesyltransferase inhibitor, PRL-1, -2, and -3 shifted into the nucleus. Furthermore, a mutant form of PRL-2 lacking the C-terminal prenylation signal is associated with the nucleus. These results establish that the primary association of PRL-1, -2, and -3 with the membrane of the cell surface and the early endosome is dependent on their prenylation and that nuclear localization of these proteins may be triggered by a regulatory event that inhibits their prenylation.
Highlights
The PRL phosphatases (PRL-1, -2, and -3) are three closely related intracellular enzymes that possess the protein-tyrosine phosphatase (PTP) active site signature sequence CX5R (4 – 8)
When geranylgeranyl pyrophosphate was used as the isoprenoid donor, PRL-1 and -2 were geranylgeranylated, but PRL-3 was not (Fig. 1)
In contrast to PRL-1, PRL-3 was still not modified by geranylgeranylation (Fig. 1B, top panel), confirming that even when efficiently farnesylated, PRL-3 is not an in vitro substrate of a GGT
Summary
Materials—CHO-K1 and other cell lines were obtained from the American Type Culture Collection (Manassas, VA). Expression of Recombinant Proteins in Bacteria—To construct plasmids expressing wild-type PRLs, the coding regions of PRL-1, -2, and -3 were retrieved by PCR using Pfu polymerase and appropriate forward primers incorporating a BamHI or a SalI restriction site and reverse primers incorporating an EcoRI restriction site. Mutant PRL-2 (C101S), where the essential cysteine residue of the active site of the phosphatase was replaced by serine, was obtained through a first PCR reaction using a forward primer corresponding to the desired nucleotide substitution (5Ј-TGTGTTGCAGTGCATAGTGTTGCAGGATTGGGA-3Ј), and the reverse primer was used to amplify wild-type PRL-2. The completed first PCR reaction was used as a template in a second PCR reaction employing the forward (with an added BamHI site) and reverse (with an added EcoRI site) primers used to amplify wild-type PRL-2 (above).
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