Binding of phosphatase-1 δ to the retinoblastoma protein pRb involves domains that include substrate recognition residues and a pRB binding motif

Protein Ser/Thr phosphatase-1 (PP1) controls the retinoblastoma protein (pRb) function, including its dephosphorylation at mitotic exit. Since PP1 delta was found to coimmunoprecipitate with pRb from mitotic and early G1 cells, we further investigated the PP1 delta -pRb association using GST-full length and GST-deletion mutants of delta. GST-delta pulled-down pRb from G2, mitotic and G1 HeLa cells, thus confirming the coimmunoprecipitation results, Among the 6 deletion mutants tested, pRb was pulled down by mutant 159-295, which reproduces the C-terminal domain of 6 without the C-terminus, whereas the C-terminus alone did not pull-down pRb, Further fragmentation of the 159-295 mutant indicated that pRb was pulled down by fragment 195-260, which includes several residues involved in substrate binding, and by fragment 159-212, which contains the putative pRb-binding motif LxSxE. Altogether the results supported the hypothesis that PP1 delta may contribute to the dephosphorylation of pRb at mitotic exit and that the PP1 delta -pRb interaction may be at multiple sites. (C) 2001 Academic Press.