Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI

Abstract

Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase IH, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpl cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane.

Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1

“…We have shown, by all criteria employed so far, that the BHV-1 proteins produced by PRV were indistinguishable from those made by BHV-1 virus itself: we used Western blot analysis, pulse-chase radiolabeling experiments, endoglycosidase digestion, immunofluorescence, and in vitro translation to arrive at this conclusion (data reviewed but not shown in this paper). We did not analyze phosphorylation or sulfation, modifications that have been reported or predicted for other gE-gI homologs (7,8,25,39,40). We did insert the BHV-1 gI gene into the PRV gI locus, but we found that expression was considerably less efficient (15-fold lower than that from gG).…”

A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network.

“…The second important determinant regulating gpl trafficking is the highly acidic stretch of amino acids (587-DDFEDSESTDTEEE602) that contains four hydroxylated residues (Ser593, SerS95, ThrS96, ThrS98), two of which (ThrS96 and 598) fall into consensus sites for casein kinase II phosphorylation. Previous work showed that threonines 596 and 598 can be phosphorylated in vivo and in vitro by purified casein kinase II (Yao et al, 1993b). Accordingly, the removal of two threonines leads to a considerable redistribution of gpl to the cell surface, whereas the single mutation of any one of the two threonines does not affect the steady-state distribution of the protein.…”

Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail.

“…In addition to furin, other secretory pathway membrane proteins have been argued to be substrates for CKII, including the cytoplasmic tails of the signal sequence receptor (SSR) (Ou et al, 1992), calnexin (Ou et al, 1992), synaptotagmin (p65) (Bennett et al, 1993;Davletov et al, 1993), the varicella-zoster virus Fc receptor (Yao et al, 1993), the polymeric immunoglobulin receptor (Casanova et al, 1990), and both the cation-independent and -dependent mannose 6-phosphate receptors (Meresse et al, 1990;Rosorius et al, 1993;Korner et al, 1994). The phosphorylation of the rough endoplasmic reticulum (RER)-resident proteins SSR and calnexin together with profurin ( Figure 4) argues for a RER-localized CKII activity that phosphorylates a number of membrane proteins.…”