MCL1 binding to the reverse BH3 motif of P18INK4C couples cell survival to cell proliferation

Abstract

Commitment to cell cycle entry and cellular duplication is a tightly coordinated and regulated process. Once initiated, a series of multiple checkpoints ensure both accurate genomic replication and chromosomal separation. In the event of unsuccessful cell division, parallel pathways exist that induce the cell to undergo programmed cell death, or apoptosis. At the center of such stress-induced, intrinsic apoptotic regulation lies the BCL2 family of pro-and antiapoptotic regulatory proteins. In a proliferative state the balance of pro-and anti-apoptotic signaling proteins would be expected to favor an excess population of anti-apoptotic members. While the anti-apoptotic BCL2 family member, MCL1, has been identified to oversee mitotic progression, direct communication between the BCL2 family and cell proliferation has not been observed. In this study, we demonstrate a direct protein-protein interaction between MCL1 and the G 1 /S checkpoint protein, P18INK4C. This interaction is mediated by a reverse BH3 (rBH3) motif located in P18INK4C's C-terminal ankyrin repeat. MCL1 is further shown to decrease P18INK4C expression and thereby regulate cell cycle entry in a retinoblastoma (RB1)-dependent manner. Our findings establish a mechanism for translation independent and direct communication between the BCL2 family regulation of apoptosis and CDK4/6-RB regulation of early G 1 /S transition during cellular division/growth.

MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73

“…Here, we establish a unique function of MCL1 in transcriptional regulation that has never been previously described. As this is the second account of an rBH3-mediated interaction with MCL1, our work provides further validation that the rBH3 motif is a biologically relevant and available mechanism for mediating interactions between the Bcl-2 family and other protein signaling networks 11,12 . By characterizing these rBH3-mediated interactions, we gain a better understanding of the core homeostatic mechanisms regulating MCL1.…”

“…These findings support and expand on the role that the rBH3 motif plays in extending MCL1 function in cells. Recently, we published a study showing that MCL1 directly modulates protein stability of the cell cycle regulator P18 through a homologous rBH3-mediated interaction 12 . Our work here provides further evidence that the rBH3 motif is capable of mediating interactions with MCL1 and implies other rBH3-containing proteins should be explored.…”

“…In addition to several DNA damage response studies, nuclear MCL1 has also been implicated in cell cycle regulation 12 , 37 , 59 . In 2000, Fujise and colleagues identified that MCL1 interacts with proliferating cell nuclear antigen (PCNA) in the nucleus to regulate cell cycle progression through S-phase.…”

“…To study the p73 TD , we synthesized a corresponding 23 amino acid peptide (p73 23mer ) that parallels the homologous BH3 peptides used for other Bcl-2 family binding studies 12,25,26 (Table 2). Through an N-terminally labeled FITC-p73 23mer peptide, we determined that p73 binds MCL1 with a K D that is ≤5 nM (limit of detection for our probe) (Fig.…”

“…Therefore, we refer to these sequences as having a reverse-BH3 or rBH3 motif. Using BLAST sequence analysis of the human proteome, we identified a series of native proteins that contain rBH3-like sequences, thereby establishing a pool of potential novel MCL1-binding partners 11,12 .…”