E1A modulates phosphorylation of p130 and p107 by differentially regulating the activity of G1/S cyclin/CDK complexes

Matilde Parrẽo, Judit Garriga, Ana Limón, Jeffrey H. Albrecht, Xavier Grãa

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15 Scopus citations

Abstract

We have previously shown that the adenoviral 12S E1A protein modulates the phosphorylation status of p130 and p107 without apparent changes in the cell cycle dependent phosphorylation of the retinoblastoma protein. Here we report on the mechanisms by which E1A modifies differentially the phosphorylation status of pocket proteins. In human U-2 OS osteosarcoma cells transiently expressing E1A, ectopic expression of D-type cyclins alone or combined, but not cyclins E and/or A, fully rescues E1A-mediated block in hyperphosphorylation of p130 to form 3. However, cyclins E and A, individually or together, induce hyperphosphorylation of p130 to species with intermediate mobility. Phosphopeptide maps indicate that E1A inhibits phosphorylation of sites phosphorylatable by CDKs. One of these sites is Ser-1044. The effects of blocking the activities of endogenous and exogenous cyclins with p16 and dominant negative CDK2 in E1A expressing cells further indicate that p130 is phosphorylated by both D-type cyclin and cyclin E/CDK complexes and that E1A modulates the activity of these G1/S CDKs by independent mechanisms. Stable expression of E1A in MC3T3-E1 cells leads to downregulation of D-type cyclins, and upregulation of cyclins E and A. This is accompanied by increased CDK2 kinase activity. Downregulation of D-type cyclins in these cells correlates with a block on both p130 hyperphosphorylation to form 3 and hyperphosphorylation of p107. This is rescued by D-type cyclins but not by cyclin E. In addition, we show that the upregulation of cyclins E and A is at least partially dependent on an intact pocket protein/E2F pathway, but downregulation of D-type cyclins is not. Moreover, we provide evidence that while the lack of a functional pRB pathway also results in a block on hyperphosphorylation of p130 to form 3, this is not sufficient to induce constitutive expression of p130 form 2b.

Original languageEnglish (US)
Pages (from-to)4793-4806
Number of pages14
JournalOncogene
Volume20
Issue number35
DOIs
StatePublished - Aug 9 2001

Bibliographical note

Funding Information:
We thank Thomas Young, Renée Marshall, Rashmi Kumar and Sabyasachi Battacharya for critical reading of the manuscript and Rosemary Dillon for editorial assistance. We thank George R Beck, Elizabeth Moran, Yue Xiong, Pilar Ruiz-Lozano and Ed Harlow for antibodies, plasmids, adenoviruses and cell lines. M ParrenÄ o, J Garriga and A Limón, were partially supported by fellowships from Dirección General de Investigación Cientṍ fica y Técnica (Ministerio de Educación y Cultura, Spain). This work was supported in part by grants to X GranÄ a including a grant from the National Institute of

Keywords

  • Adenovirus
  • Cyclin dependent kinase (CDK)
  • Cyclins
  • Large T antigen (TAg)
  • Retinoblastoma protein

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