Ser-634 and Ser-636 of Kaposi’s Sarcoma-Associated Herpesvirus RTA are Involved in Transactivation and are Potential Cdk9 Phosphorylation Sites

Abstract

The replication and transcription activator (RTA) of Kaposi’s sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal (527KKRK530) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that 634SPSP637 motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full transcriptional potency of K-RTA.

Discoidin Domain Receptor-1 (DDR1) is Involved in Angiolymphatic Invasion in Oral Cancer

“…After magnetic separation, the Dynabeads were washed thoroughly with washing buffer three times followed by immunocomplex elution. The cell lysates or the eluted immunocomplex was subjected to standard western blot analysis as described previously [58].…”

KSHV Reactivation and Novel Implications of Protein Isomerization on Lytic Switch Control

“…Rta protein has an apparent molecular weight of 73.7–120 kilodalton (kDa), a difference indicative of its extensive posttranslational modifications, predominantly phosphorylation (20 kDa alone) [ 7 , 34 , 37 , 42 , 43 , 44 ]. Rta is also ADP-ribosylated [ 45 ], and may contain other modifications.…”

“…Posttranslational modifications are absolutely vital to the proper function of proteins within a cell, for signaling, conformation, interactions with other factors, stability, localization, DNA binding and transactivation, among many others. A number of potential modifications to Rta include phosphorylation, sumoylation, ubiquitylation and proline-directed modifications such as prolyl hydroxylation and prolyl isomerization [ 2 , 4 , 7 , 34 , 37 , 42 , 43 , 44 , 45 , 46 , 54 , 55 , 64 , 68 , 110 ]. The demonstrated importance of prolines within Rta may not have been limited to a role in tetramerization, but could have broader consequences on Rta function.…”

P-TEFb as A Promising Therapeutic Target

“…Further studies are required to complete the list of CDK9 substrates in cells. CDK9 also phosphorylates viral proteins such as Kaposi's Sarcoma Associate Herpesvirus (KSHV) RTA proteins, which modulate viral replication [116].…”