Nuclear Localization of the Cell Cycle Regulator CDH1 and Its Regulation by Phosphorylation

Abstract

The anaphase-promoting complex activated by CDC20 and CDH1 is a major ubiquitination system that controls the destruction of cell cycle regulators. Exactly how ubiquitination is regulated in time and space is incompletely understood. Here we report on the cell cycle-dependent localization of CDH1 and its regulation by phosphorylation. CDH1 localizes dynamically to the nucleus during interphase and to the centrosome during metaphase and anaphase. The nuclear accumulation of CDH1 correlates with a reduction in the steady-state amount of cyclin A, but not of cyclin E. A nuclear localization signal conserved in various species was identified in CDH1, and it sufficiently targets green fluorescent protein to the nucleus. Interestingly, a CDH1-4D mutant mimicking the hyperphosphorylated form was constitutively found in the cytoplasm. In further support of the notion that phosphorylation inhibits nuclear import, the nuclear localization signal of CDH1 with two phospho-accepting serine/threonine residues changed into aspartates was unable to drive heterologous protein into the nucleus. On the other hand, abolition of the cyclin-binding ability of CDH1 has no influence on its nuclear localization. Taken together, our findings document the phosphorylation-dependent localization of CDH1 in vertebrate cells.The timely and orderly progression of eukaryotic cell division cycle is precisely controlled through ubiquitination-induced proteolysis. The anaphase-promoting complex (APC) 1 functions as a major ubiquitin ligase in the cell, and it governs anaphase onset, mitotic exit, and G 1 events (1-4). APC is a large complex of multiple subunits. It catalyzes the formation of polyubiquitin chains on cyclins and other cell cycle regulators and thereby targets them for degradation by proteosome. The components of APC are highly conserved in organisms ranging from yeast to humans. The rise and fall of APC activity is stringently regulated in a cell cycle-dependent manner. It plateaus at the transition from metaphase to anaphase, remains high throughout G 1 , and drops in S/G 2 until early mitosis (5). One important mechanism for the regulation of APC is through phosphorylation of its subunits (6 -9). In addition, CDC20 and CDH1 can act as activators and specificity factors of APC (10 -15).CDC20 and CDH1 are WD40 repeat proteins that associate transiently with and activate APC in a substrate-specific fashion. Many substrate proteins targeted by the CDC20-and CDH1-activated APC share a sequence motif known as destruction box (5, 16). Another motif termed the KEN box is recognized by CDH1-activated APC and has been found in substrates including CDC20, NEK2, HSL1, mitotic cyclins, and securin (17)(18)(19)(20). In addition, a distinct recognition domain called the A box has been shown to be required for CDH1-dependent ubiquitination and degradation of the Aurora-A kinase (21). Exactly how CDC20 and CDH1 activate APC remains to be fully understood. Existing evidence suggests that they mediate the temporal and spatial activation of APC throu...

APC/C CCS52A complexes control meristem maintenance in the Arabidopsis root

“…were nuclear, which corresponded to the reported active form of the yeast and human orthologues (23,24).…”

Anaphase-promoting complex-dependent proteolysis of cell cycle regulators and genomic instability of cancer cells

“…However, there are also reports that Cdh1 levels oscillate during the cell cycle at least in human cells Hsu et al, 2002). The subcellular localization of Cdh1 is cell cycle regulated (Jaquenoud et al, 2002;Zhou et al, 2003) and Cdh1 is nuclear during G1 and in the cytoplasm between S phase and the end of mitosis (Jaquenoud et al, 2002). Cdkdependent phosphorylation leads to efficient inactivation of Cdh1 by nuclear export (Jaquenoud et al, 2002) and prevention of APC binding Jaspersen et al, 1999;Lukas et al, 1999;Kramer et al, 2000), thus restricting APC Cdh1 activity to the points in the cell cycle when Cdk activity is low.…”

Nuclear translocation of Skp2 facilitates its destruction in response to TGFβ signaling

“…Previous work suggested that the cellular presence of Cdh1 is in the nucleus. 21,22 Localization of Skp2 has been variable depending on cell type. [23][24][25][26][27] To assess the localization of Cdh1 and Skp2 in Mv1Lu cell, we performed immunocytochemistry in the presence and absence of TGFβ.…”