Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb.
Single cell analysis allows high resolution investigation of temporal relationships between transition events in G1. It has been suggested that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is the molecular mechanism behind passage through the restriction point (R). We performed a detailed single cell study of the temporal relationship between R and pRb phosphorylation in human fibroblasts using time lapse video-microscopy combined with immunocytochemistry. Four principally different criteria for pRb phosphorylation were used, namely (i) phosphorylation of residues Ser795 and Ser780, (ii) degree of pRb-association with the nuclear structure, a property that is closely related with pRb phosphorylation status, (iii) release of the transcription factor E2F-1 from pRb, and (iv) accumulation of cyclin E, which is dependent on phosphorylation of pRb. The analyses of individual cells revealed that passage through R preceded phosphorylation of pRb, which occurs in a gradually increasing proportion of cells in late G1. Our data clearly suggest that pRb phosphorylation is not the molecular mechanism behind the passage through R. The restriction point and phosphorylation of pRb thus seem to represent two separate check point in G1.
Martinsson
Hanna-Stina
HS
Department of Oncology-Pathology, Karolinska Institutet, CCK R8:04, KS 171 76 Stockholm, Sweden.
Starborg
Maria
M
Erlandsson
Fredrik
F
Zetterberg
Anders
A
D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D056741:Cyclin D; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases / Q000502:physiology; D016213:Cyclins / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005347:Fibroblasts / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000502:physiology; D016160:Retinoblastoma Protein / Q000032:analysis / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D018797:Cell Cycle Proteins; D056741:Cyclin D; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases