NMR and mass spectrometry studies of putative interactions of cell cycle proteins pRb and CDK6 with cell differentiation proteins MyoD and ID-2.
Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein-protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.
Smialowski
Pawel
P
Max Planck Institute for Biochemistry, 82152 Martinsried, Germany.
Singh
Mahavir
M
Mikolajka
Aleksandra
A
Majumdar
Sudipta
S
Joy
Joma K
JK
Nalabothula
Narasimharao
N
Krajewski
Marcin
M
Degenkolbe
Roland
R
Bernard
Hans-Ulrich
HU
Holak
Tad A
TA
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002645:Chickens; D051361:Cyclin-Dependent Kinase 6; D050764:Cyclin-Dependent Kinase Inhibitor p19; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; C493974:CDKN2D protein, human; D018797:Cell Cycle Proteins; D050764:Cyclin-Dependent Kinase Inhibitor p19; D004268:DNA-Binding Proteins; C497450:ID2 protein, human; D051797:Inhibitor of Differentiation Protein 2; D017570:MyoD Protein; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; C059731:oncogene protein E7, Human papillomavirus type 16; D004247:DNA; C495911:CDK6 protein, human; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases