title: Genetic analysis of the polyomavirus DnaJ domain.
abstract: Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60(c-src) and in transformation.
authors: Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, USA.
authors: Kerry A
authors: KA
authors: Whalen
authors: Rowena
authors: R
authors: de Jesus
authors: Jennifer A
authors: JA
authors: Kean
authors: Brian S
authors: BS
authors: Schaffhausen
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011120:Polyomavirus / Q000235:genetics* / Q000378:metabolism; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology; D050717:Retinoblastoma-Like Protein p130; D015533:Transcriptional Activation
chemical_list: D000957:Antigens, Viral, Tumor; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C494372:Hspa8 protein, mouse; D011506:Proteins; C493837:RBL2 protein, human; C493838:Rbl2 protein, mouse; D050717:Retinoblastoma-Like Protein p130; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000251:Adenosine Triphosphatases