Nuclear Bmp4 interacts with the SCF E3 ubiquitin ligase complex and inhibits the cell cycle

Abstract

A variant of BMP‐4, called nBmp4, is translated from a downstream alternative start codon and localized to the nucleus. A yeast two‐hybrid screen identified the RING finger protein Regulator of Cullins 1 (ROC1) as a binding partner of nBmp4. ROC1 is a component of several different SCF E3 ubiquitin ligase complexes, and co‐immunoprecipitation experiments revealed that nBmp4 also interacts with several other SCF E3 ligase components including ROC2, Cullins 1, 2, 3, 4A, and 5, and F‐box proteins Skp2 and beta‐TrCP1. Overexpression of nBmp4 in HEK293T cells in culture caused cells to accumulate in G0/G1 phase of the cell cycle (as measured by DNA content) and increased the number of small cells in the population (as measured by forward scatter in a flow cytometer), suggesting that nBmp4 inhibits the cell cycle and may lead to cell death. Two of the known targets of ubiquitination by ROC1‐containing SCF E3 ligase complexes are the cell cycle inhibitors p21 and p27, and targeted inactivation of ROC1 causes p27 to accumulate, inhibiting embryonic cell division. To determine whether nBmp4 similarly causes p27 and/or p21 to accumulate by interfering with ROC1 function, nBmp4 was overexpressed in cultured cells and protein isolates were analyzed by immunoblotting. Neither p27 nor p21 levels were elevated, suggesting that nBmp4 inhibits the cell cycle by a different mechanism. Funding was provided by the NIH #AR048839.