HSP27 Multimerization Mediated by Phosphorylation-sensitive Intermolecular Interactions at the Amino Terminus

Abstract

. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal ␣-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg 5 -Tyr 23 , which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the ␣-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain.

Differential regulation of HSP27 oligomerization in tumor cells grown in vitro and in vivo

“…The largest molecules with a molecular mass of 600 ± 800 kDa could correspond to about 24 HSP27 subunits (Rogalla et al, 1999). It has been reported that the structure of HSP27 was highly dynamic with small oligomers being in equilibrium with bigger ones (Rogalla et al, 1999;Lambert et al, 1999). This equilibrium can be shifted toward smaller oligomers as a consequence of HSP27 serine phosphorylation.…”

“…Human HSP27 contains three sites of phosphorylation at serine residues. The serine 82 corresponds to rodent serine 90 whose phosphorylation was shown to be essential in the reduction of HSP27 complexes to small oligomers (Lambert et al, 1999). This serine is located in a highly variable region that connects the Cterminal a-crystallin domain of the molecule to its Nterminal domains (De Jong et al, 1988).…”

“…This serine is located in a highly variable region that connects the Cterminal a-crystallin domain of the molecule to its Nterminal domains (De Jong et al, 1988). It was suggested that the phosphorylation of this serine could destabilize the HSP27 multimers by modifying interactions between some domains of the monomers (Lambert et al, 1999). The in¯uence of the phosphorylation of serine 15, which is conserved between human and rodent HSP27, on the structure of the protein is more controversial (Rogalla et al, 1999;Lambert et al, 1999).…”

“…The in¯uence of the phosphorylation of serine 15, which is conserved between human and rodent HSP27, on the structure of the protein is more controversial (Rogalla et al, 1999;Lambert et al, 1999). It was suggested that phosphorylation of serine 15 could modulate interactions of HSP27 with other proteins such as protein kinase B, F-actin or granzyme A (Miron et al, 1991;Benndorf et al, 1994;Konishi et al, 1997;Beresford et al, 1998) rather than the size of HSP27 oligomers (Lambert et al, 1999). The third serine residue in human HSP27 is 1 mM).…”

Small heat-shock proteins: important players in regulating cellular proteostasis

“…Phosphorylation reduces the average oligomer size, and increases oligomeric polydispersity and rate of subunit exchange of αBc [98,[132][133][134], whereas it leads to a dramatic decrease in the size of Hsp27 oligomers, such that the triply phosphorylated isoform is predominately dimeric in solution [135,136]. Thus, under stress conditions, Hsp27 is phosphorylated, triggering dissociation of the high molecular mass Hsp27 oligomers [117,135,137,138] and an increase in the amount of exposed hydrophobicity on the newly formed Hsp27 dimers [139]. Phosphorylation also affects the cellular distribution of some sHsps.…”