Abstract
The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.
Highlights
The eukaryotic cell division cycle is tightly regulated by the activation and deactivation of the cyclin-dependent kinases (CDKs).1 Active CDK serves as a protein kinase subunit, the kinase activity of which is dependent on its association with a regulatory cyclin subunit [1,2,3]
Identification of Cyclin D3 as p58PITSLRE Protein Kinaseinteracting Protein—To identify proteins that interact with p58PITSLRE, the yeast two-hybrid system was employed with p58PITSLRE-fused LexA DNA binding domain as bait
Expression of p58PITSLRE is G2/M phase-specific, resulting from translation controlled by an internal ribosome entry site [29]
Summary
Cells were washed three times with ice-cold PBS and solubilized with 1 ml of lysis buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2, 60 mM -glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). For checking the efficiency of the immunodepletion, the precipitates were boiled in SDS sample buffer, resolved on a 10% SDS-PAGE gel, and immunoblotted with an anti-cyclin D3 antibody. Immunofluorescence—The 7721 cells were plated onto coverslips the day before synchronization After synchronization, they were fixed in ice-cold methanol for 1 h and blocked in PBS containing 10% normal blocking serum followed by an overnight reaction with the primary antibody at 4 °C. After 48 h, the transfected cells were fixed for 30 min with 3% paraformaldehyde in PBS and observed under the Leica confocal microscope as described above
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