Analysis of site-specific phosphorylation of the retinoblastoma protein during cell cycle progression

Differential phosphorylation of the retinoblastoma protein plays a pivotal role in cell cycle regulation. The retinoblastoma protein is specifically p hosphorylated during the cell cycle by cyclin-dependent kinase complexes wh ich intersect with many cellular signaling networks. Since the loss of the retinoblastoma signaling pathways occurs in a wide variety of human tumors, understanding the significance of site-specific phosphorylation can clarif y the role of selected cyclin-dependent kinase complexes during cell cycle progression. Here we describe the phosphospecificity and cellular character ization of a panel of polyclonal antibodies that recognize unique phosphory lation sites within the retinoblastoma protein. These reagents were used to validate authentic cellular retinoblastoma phosphorylation sites at amino acids 780, 795, and 807/811. correlating with the G1-S transition. (C) 1999 Academic Press.