Binding of phosphatase-1 delta 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 PP1delta was found to coimmunoprecipitate with pRb from mitotic and early G1 cells, we further investigated the PP1delta-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 delta deletion mutants tested, pRb was pulled down by mutant 159-295, which reproduces the C-terminal domain of delta 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 PP1delta may contribute to the dephosphorylation of pRb at mitotic exit and that the PP1delta-pRb interaction may be at multiple sites.
Bianchi
M
M
Department of Experimental Pathology, University of Pisa, via Roma 55, 56126 Pisa, Italy.
Villa-Moruzzi
E
E
D001665:Binding Sites; D016193:G1 Phase; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection
D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1