B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

Girish Jayadeva,Alison Kurimchak,Judit Garriga,Elena Sotillo,Anthony J Davis,Dale S Haines,Marc Mumby,Xavier Graña

doi:10.1074/jbc.m110.162354

Abstract

B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

Highlights

The retinoblastoma family of growth suppressor proteins, designated pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130
Pocket proteins remain hyperphosphorylated through the S and G2 phases and part of mitosis as they become targets of cyclin-cyclin-dependent kinase (CDK) complexes that operate at these cell cycle stages
As we have shown that expression of GST pulldown assays using these mutants revealed that the Simian virus 40 (SV40) st in exponentially growing U-2 OS cells delays dephosspacer region of p107 is essential for binding to PP2A/A and phorylation of pocket proteins when CDK activity is compro

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Treatments—Human osteosarcoma U-2 OS cells and human glioblastoma T98G cells were maintained in Dulbecco’s modified Eagles’ medium (DMEM) (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Gemini). In vitro binding assays were performed by incubating 2 ␮g of GST fusion proteins loaded onto glutathione beads with 300 ␮g of whole lysate or 1 ␮g of recombinant purified PP2A holoenzyme complexes in complete DIP buffer. In Vitro Phosphatase Assays—Soluble histone H1 and GSTp107 loaded on glutathione beads were phosphorylated with purified cyclin A-CDK2 (Millipore) in kinase buffer (50 mM HEPES (pH 7.2), 10 mM MgCl2, 5 mM MnCl2) supplemented with 50 ␮M ATP and 10 ␮Ci of [␥-32P]ATP for 1 h at 30 °C. The construct lacking the N terminus (ϪN) was generated by digesting the mutant pGEX-2T-p107 (254 –1068) and pGEX-2T-p107 pocket [385–949] with EcoRI and inserting the C terminus next to the pocket. Expression vectors were generated by PCR amplification using pGEX-2Tp107, -p130, and -pRB as templates and the primers listed in supplemental Table 2, which contain BamHI or NotI restriction enzyme sites.

The following retroviral constructs were obtained from Open

Findings

DISCUSSION