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 protein⅐protein interaction regions were mapped to the hic-5 LIM domains and the hsp27 C-terminal domain. Initial analysis of the functional role of hsp27⅐hic-5 interaction revealed that hic-5 significantly inhibited the protection against heatinduced cell death conferred by hsp27 overexpression in co-transfected 293T cells. In contrast, when a non-hsp27-interacting hic-5 truncation mutant (hic-5/⌬LIM4) 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.

Regulation of contractility by Hsp27 and Hic-5 in rat mesenteric small arteries

“…In addition, Hic-5 has been shown to bind Hsp27 (24) in rat glomerular cells, although whether such an interaction occurs in smooth muscle has not been explored. Here we show that in nonstimulated arteries, there was weak interaction between Hsp27 and Hic-5; however, NE induced a strong association between the two, an interaction that required serine phosphorylation of Hsp27 and tyrosine phosphorylation of Hic-5.…”

Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

“…The yeast-two hybrid screen was performed as previously described (Jia et al 2001). Using pGal4-BD-Hsp27 as the bait, a rat ortholog of the Arp2/3 complex protein p41-ArpC1a was identified as an Hsp27 binding protein (accession #AF315378) (Li et al 2003;Thornton et al 2006).…”

Hic-5 mediates endothelial sprout initiation by regulating a key surface metalloproteinase

“…The N-terminal LD domains of Hic-5 interact with focal adhesion kinase (FAK, also known as PTK2) (Fujita et al, 1998) vinculin (Deakin et al, 2012), paxillin (Deakin et al, 2012), c-Src kinase (Csk) (Thomas et al, 1999), protein tyrosine kinase 2 β (PYK2, also known as PTK2B) (Matsuya et al, 1998) and Arf GAP1 (GIT1) (Nishiya et al, 2002). The LIM domains at the C-terminus of Hic-5 mediate binding to PTP-PEST (also known as PTPN12) (Nishiya et al, 1999) and Hsp27 (also known as HSPB1) (Jia et al, 2001). Oxidative stress induces Hic-5 translocation into the nucleus, where it acts as a transcriptional coactivator (Shibanuma et al, 2002(Shibanuma et al, , 2003(Shibanuma et al, , 2004.…”