Physical and Functional Interaction between Myeloid Cell Leukemia 1 Protein (MCL1) and Fortilin

Abstract

Myeloid cell leukemia 1 protein (MCL1) is an anti-apoptotic protein that is structurally related to Bcl-2. Unlike other Bcl-2 family proteins that are constitutively expressed, MCL1 is inducibly expressed in cells that are recently exposed to growth and differentiation stimuli. Here, we report the identification of fortilin as a novel MCL1-interacting protein by screening of a yeast two-hybrid library with MCL1 as bait. Fortilin specifically interacted with MCL1 both in vitro andin vivo. The intracellular localization of fortilin was predominantly nuclear and identical to that of MCL1, as shown by immunostaining and confocal microscopy analysis. Fortilin, like MCL1, was rapidly inducible in serum-stimulated human aortic vascular smooth muscle cells. Although the depletion of intracellular fortilin by small interfering RNA (siRNA) against fortilin (siRNA-fortilin) did not affect intracellular MCL1 level, the depletion of intracellular MCL1 by siRNA-MCL1 was associated with the significant reduction of the fortilin protein level, without affecting the fortilin transcript numbers. In addition, a pulse-chase experiment showed that the depletion of MCL1 by siRNA-MCL1 was associated with the rapid degradation of fortilin protein, which was found quite stable in the presence of MCL1. Furthermore, the half-life of fortilin_R21A, a point mutant of fortilin lacking the binding to MCL1, was significantly shorter than that of wild-type fortilin as shown by a pulse-chase experiment. These data suggest that MCL1, in addition to being an anti-apoptotic molecule, serves as a chaperone of fortilin, binding and stabilizing fortilin in vivo. Taken together with our previous observation that fortilin overexpression prevents cells from undergoing apoptosis (Li, F., Zhang, D., and Fujise, K. (2001) J. Biol. Chem. 276, 47542–47549), it is likely that MCL1, an anti-apoptotic protein inducible by growth and differentiation stimuli, stabilizes another anti-apoptotic protein fortilin maximizing the prosurvival environment in cells.