Figure 2 - Polyubiquitination of p27^Kip1 by KPC.

(a) Purified recombinant human His₆–Flag–KPC1 and His₆–HSV–KPC2 expressed in insect cells were subjected to SDS–PAGE and staining with Coomassie blue. (b) Interaction between KPC1 and KPC2 in insect cells. Lysates of cells expressing the indicated combinations of KPC2 and either wild-type (WT) KPC1 or the KPC1(R) mutant were subjected to immunoprecipitation (IP) with anti-Flag, and the resulting precipitates were subjected to immunoblot analysis (IB) with anti-Flag or anti-HSV. (c–e) Polyubiquitination of p27^Kip1 mediated by recombinant KPC. The recombinant KPC1–KPC2 complex was assayed for the ability to mediate polyubiquitination of p27^Kip1, p57^Kip2, or p21^Cip1 in the presence of the indicated combinations of Uba1, 100 ng (c) or 30 ng (d, e) of UbcH5A, GST–ubiquitin (c, d) or ubiquitin (e), and ATP. (f) Requirement for the RING-finger domain in KPC1 function. KPC1 derivatives (WT or R) were assayed for the ability to mediate polyubiquitination of p27^Kip1 in the absence or presence of KPC2. (g) E2 preference of KPC. The indicated E2 enzymes were tested for their ability to support the polyubiquitination of p27^Kip1 by recombinant KPC1–KPC2.