Identification and Characterization of hic-5/ARA55 as an hsp27 Binding Protein

Abstract

hsp27 has been reported to participate in a wide variety of activities, including resistance to thermal and metabolic stress, regulation of growth and differentiation, and acting as a molecular chaperone or a regulator of actin polymerization. We hypothesized that these diverse functions are regulated in a cell- or tissue-specific manner via interaction with various binding proteins. To investigate this hypothesis, we used hsp27 as a "bait" to screen a yeast two-hybrid cDNA library from rat kidney glomeruli and identified a novel hsp27 binding protein, hic-5 (also known as ARA55), a focal adhesion protein and steroid receptor co-activator. Biochemical interaction between hsp27 and hic-5 was confirmed by co-immunoprecipitation, and critical protein· protein interaction regions were mapped to the hic-5 LIM domains and the hsp27 C-terminal domain. Initial analysis of the functional role of hsp27·hic-5 interaction revealed that hic-5 significantly inhibited the protection against heat-induced cell death conferred by hsp27 overexpression in co-transfected 293T cells. In contrast, when a non-hsp27-interacting hic-5 truncation mutant (hic-5/ΔLIM4) was co-expressed with hsp27, the hic-5 inhibition of hsp27 protection was absent. We conclude that hic-5 is a true hsp27 binding protein and inhibits the ability of hsp27 to provide protection against heat shock in an interaction-dependent manner.