BIOLOGICAL OVERVIEW

Heat shock proteins (HSPs) serve multiple functions in a variety of organisms as evolutionarily distant as bacteria and humans. In the fly, there are many genes coding for multiple families of HSPs that function in regulatory pathways, both developmental and the response to heat stress. The developmental regulation is consonant with a role of HSPs as molecular chaperones. They are involved in protein folding: they aid in the establishment of native three dimensional structure for a variety of proteins that require cellular functions in order to attain such structure. HSPs function as partners for various signal transducing proteins, whose signaling dynamics are regulated by association with HSPs (Morimoto, 1994).

Organisms respond to elevated temperatures by rapidly inducing HSPs. The regulation of this response is mediated at the transcriptional level by a transcription factor called heat shock factor (HSF), which binds to the regulatory shock elements that are present upstream of all heat shock genes. Thus, HSF responds to the heat shock signal by binding to DNA and regulation of target gene transcriptional competence.

Stress induced binding of HSF to DNA is controlled by the transition of HSF from a monomer to a trimer. Trimerization increases the affinity for the heat shock elements of HSPs by several orders of magnitude, through the assembly of one trimeric protein complex containing three DNA-binding domains, one contributed from each HSF monomer (Wu, 1995).

How does the organism at the cellular level recognize heat shock duress and induce activation of heat shock factor? One idea is that a physical interaction between HSF and HSPs suppresses induction of the DNA-binding of HSF. HSPs may assist in constraining HSF in a latent, monomeric state. When the organism is subjected to heat stress, multiple protein species denature, increasing the demand on HSPs to refold proteins, thereby releasing HSF from cytoplasmic constraint, and allowing its transportation to the nucleus where it undergoes trimerization, and subsequent binding to the promoters of HSPs.

Although HSF associates with HSPs in vitro, and the interaction of HSF and hsp70 (Heat shock protein with a molecular weight of 70 kDa) has been documented, interaction of hsp70 with HSF does not correlate with the level of activation of HSF. Both latent and active forms of HSF interact with hsp70. Elevated concentrations of hsp70 alone do not significantly inhibit induction of the DNA-binding activity of endogenous HSF, and elevated concentrations of hsp70 do not alter the induction of HSPs (Rabindran, 1994).

Some clarification of the pathway followed during HSF activation comes from the study of the human HSF1 protein. A region of HSF1, located between the leucine repeat trimerization domain and the C-terminal transactivation domain functions as a regulatory domain. By itself, the regulatory domain can confer heat shock inducibility to chimeric proteins containing the activation domain, but it fails to function as a DNA binding domain. This function is served by the N-terminal sequence (Green, 1995).

Subsequent studies have shown that the regulatory domain of HSF1 is sufficient to sense heat stress. The regulatory domain can even function with a heterologous transcriptional activation domain, suggesting that the regulatory domain can function irrespective of the sequence of the C-terminal transactivation domain (Newton, 1996).

Phosphorylation has been thought of as a mechanism for modulation of heat shock factor activity. Phosphorylation of transcription factors can occur through a number of different pathways in the cell. For example, the Raf/ERK (extracellular-signal-regulated kinase) pathway is activated by FGF-receptor and EGF-receptor in Drosophila (See Rolled). Another example is the MEKK/SAP (JNK) (stress activated protein kinase/Jun N-terminal kinase) pathway, found in both mammals and Drosophila. Hemipterous and Basket/JNK are components of this pathway in Drosophila. In humans, phosphorylation serves to repress HSF 1 at control temperatures. Two specific proline-directed serine motifs in the regulatory domain are important for phosphorylation to function normally. Stimulation of the Raf/ERK pathway results in an increased level of phosphorylation at these motifs. The regulatory domain is an excellent substrate for the mitogen-activated MAPK/ERK. It has been concluded that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1, and it is thought that this modification serves to control the response of the cell to growth control signals (Knauf, 1996).

Although such a mechanism may not function in Drosophila, its existence in humans points to the complex forces acting on HSF and its function. Studies of transcriptional activation by HSF in Drosophila have yielded insight into the process of chromatin remodeling required for gene activation (Tsukiyama, 1995a and b). This work is discussed in greater detail at the ISWI site.

GENE STRUCTURE

Bases in 5' UTR - 228

Bases in 3' UTR - 445

PROTEIN STRUCTURE

Amino Acids - 691

Structural Domains

The human and Drosophila heat shock transcription factors (HSFs) are multi-zipper proteins with high-affinity binding to DNA, regulated by heat shock-induced trimerization. Formation of HSF trimers is dependent on hydrophobic heptad repeats located in the amino-terminal region of the protein. Two subregions at the carboxyl-terminal end of human HSF1 maintain the monomeric form of the protein under normal conditions. One of these contains a leucine zipper motif that is conserved between vertebrate and insect HSFs. This suggests that the carboxyl-terminal zipper may suppress formation of trimers by the amino-terminal HSF zipper elements by means of intramolecular coiled-coil interactions that are sensitive to heat shock (Rabindran, 1993).

The HSF is a multidomain protein consisting of an N-terminal DNA binding domain. Adjacent to this domain is a hydrophobic heptad repeat (leucine zipper) region involved in HSF trimerization. There is an additional heptad repeat region just N-terminal to the C-terminal activation domain. The DNA binding and heptad repeat regions are both conserved. Conserved regions could be sites for heat shock-induced phosphorylation (Clos, 1990). It has beensuggested that the N- and C-terminal heptad repeats self associate in the monomer, contributing to its stability in non-heat shocked cells. Deletion of the central region, between the N- and C-terminal hydrophobic heptad repeat regions, results in a protein that shows no inducible changes in the native size under heat shock conditions. Short deletions in this region suggest that many of its functions are redundant. Such results suggest that the internal region of HSF can contribute to the stability of the monomer. The internal region contains a nuclear localization sequence. C-terminal to the DNA binding domain is a region important for either the proper maintenance of monomer structure or for repression of trimer formation under non-heat shock conditions. The C-terminal activator domain may contribute to the regulation of HSF oligomerization (Orosz, 1996).

The solution structure of the DNA-binding domain of the Drosophila heat shock transcription factor resembles that of the helix-turn-helix class of DNA-binding proteins. The domain comprises a four-stranded antiparallel beta-sheet, packed against a three-helix bundle. The second helix is significantly distorted and separated from the third helix by an extended turn that is subject to conformational averaging on an intermediate time scale. Helix 3 forms a classical amphipathic helix with polar and charged residues exposed to the solvent. Upon titration with DNA, resonance shifts in the backbone and Asn and Gln side-chain amides indicate that helix 3 acts as the recognition helix of the heat shock transcription factor (Vuister, 1994).

The transactivation function of HSF is conferred by the extreme C-terminal region of the protein. Deletion analysis of HSF fragments fused to the GAL4 DNA-binding domain demonstrates that transactivation is dependent on HSF residues 610-691. This domain is located beyond the C-terminal heptad repeat (leucine zipper 4) whose presence or integrity is dispensable for transactivation. The transactivation domain is functional in the absence of heat shock and can be replaced by the extreme C-terminal region of human HSF1. The Drosophila and human HSF transactivation domains are both rich in hydrophobic and acidic residues and may be structurally conserved, despite limited sequence identity (Wisniewski, 1996).

date revised: 27 Jan 97 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.