Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

Self Cite

122

The varicella-zoster virus (VZV) genome contains 70 reading frames (ORF), 5 of which encode the glycoproteins gpl, gpII, gpIII, gprV, and gpV. ORF 67 and 68 lie adjacent to each other in the unique short region of the VZV genome and code for gpIV and gpl, respectively. These two genes, which are contained within the HindIII C fragment of the VZV genome, were subcloned in the correct orientation downstream from the promoter regions of the eukaryotic expression vectors pCMV5 and pBJ. After transfection, 5 to 20% of the Cos cells bound antibody specific for the given glycoprotein. In this study, it was shown that only the cells transfected with the gpI construct bound to the Fc fragment of human immunoglobulin G. Neither the transfected gpIV gene product nor the vector only bound to the Fc fragment. Thus, VZV gpI is confirmed to be the VZV-encoded Fc-binding glycoprotein. Like the wild-type form of gpI expressed in VZV-infected cells, gpl precipitated from transfected cells contained both N-linked and 0-linked glycans and was heavily sialated. In addition, the transfected gpl gene product was phosphorylated both in cell culture and in protein kinase assays by mammalian casein kinases I and II. Extensive computer-assisted analyses of the VZV gpl sequence, as well as those of alphaherpesviral homolog glycoproteins, disclosed properties similar to those of other cell surface receptors; these included (i) exocytoplasmic regions rich in cysteine residues, (ii) membrane-proximal regions with potential 0-linked glycosylation sites, and (iii) cytoplasmic domains with consensus phosphorylation sites.

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Receptor properties of two varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpes simplex virus gE and gI

Litwin

Jackson

Grose

1992

Self Cite

122

“…Based on the results in this report, we postulate an intriguing network of phosphorylation interactions between the two components of the VZV gE:gI complex. The cytoplasmic tail of the gE constituent of this complex is phosphorylated by CKII at threonines and serines between amino acid residues 593 and 598; in addition, CKII binds to and coprecipitates with gE (17,41,58). CKII is normally active in mammalian cells, and the phosphorylation sites are not susceptible to dephosphorylation activity (54).…”

Section: Discussionmentioning

confidence: 99%

Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase

Ye¹,

Duus

Peng

et al. 1999

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.

“…A growing number of VZV proteins are now recognized as phosphoproteins, including glycoproteins I and IV (9), the putative protein kinases 47 and 66 (19,32), and the potential regulatory proteins 61 (31), 62 (7,20), 63 (20,33), and 4 (20). Some of the protein kinases involved in these modifications have been identified: casein kinase I modifies gpI in vitro (9), 47 modifies 62 in vitro (20), and CKII has previously been shown to phosphorylate gpI (9) and 62 (20). We now add 63 to the list of VZV proteins modified by CKII, at least in vitro.…”

mentioning

confidence: 99%

Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein

Stevenson

Xue

Hay

et al. 1996

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63⌬N) or 1 to 210 (63⌬K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63⌬KF fusion proteins in vitro but did not phosphorylate the 63⌬NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35 S-or 32 P-labelled extracts of cells transfected with plasmids expressing 63, 63⌬N, or 63⌬K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63⌬K and 63⌬N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization.