Member Of Heat Shock Protein Family Research Articles

Lens Crystallins and Their Microbial Homologs: Structure, Stability, and Function

Referee: Franz Schmid, Biochemicshes Laboratorium, Universitaet Bayeuth, D-95440 Bayeuth, Germanyabg-Crystallins are the major protein components in the vertebrate eye lens — a as a molecular chaperone and b and g as structural proteins. Surprisingly, the latter two share some structural characteristics with a number of microbial stress proteins. The common denominator is not only the Greek key topology of their polypeptide chains but also their high intrinsic stability, which, in certain microbial crystallin homologs, is further enhanced by high-affinity Ca2+-binding. Recent studies of natural and mutant vertebrate bg-crystallins as well as spherulin 3a from Physarum polycephalum and Protein S from Myxococcus xanthus allowed the correlation of structure and stability of crystallins to be elucidated in some detail. From the thermo-dynamic point of view, stability increments come from (1) local interactions involved in the close packing of the cooperative units, (2) the all-b secondary structure of the Greek-key motif, (3) intramolecular interactions between domains, (4) intermolecular domain interactions, including 3D domain swapping and (v) excluded volume effects due to “molecular crowding” at the high cellular protein concentrations. Apart from these contributions to the Gibbs free energy of stability, significant kinetic stabilization originates from the high activation energy barrier determining the rate of unfolding from the native to the unfolded state. From the functional point of view, the high stability is responsible for the long-term transparency of the eye lens, on the one hand, and the stress resistance of the microorganisms in their dormant state on the other. Local structural perturbations due to chemical modification, wrong protein interactions, or other irreversible processes may lead to protein aggregation. A leading cataract hypothesis is that only after a-crystallin, a member of the small heat-shock protein family, is titrated out does pathological opacity occur. Understanding the structural basis of protein stability in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.

Heat acclimation increases the basal HSP72 level and alters its production dynamics during heat stress.

It has been previously shown that heat acclimation leads to an elevated basal level of 72-kDa heat shock protein (HSP72). Augmented expression of HSP72 is considered as a cytoprotective response. This led us to hypothesize that alterations in the heat shock protein (HSP) defense pathway are an integral part of the heat acclimation repertoire. To investigate this, we studied the temporal profile of basal HSP expression upon acclimation and the dynamics of their accumulation subsequent to acute heat stress (HS). In parallel, HSP72 mRNA level before and after HS was measured. For comparison, HSC mRNA [the constitutive member of 70-kDa HSP (HSP70) family] was measured in similar conditions. Heat acclimation was attained by continuous exposure of rats to 34 degrees C for 0, 1, 2, and 30 days. HS was attained by exposure to 41 or 43 degrees C for 2 h. Thermoregulatory capacity of the rats was defined by rectal temperature, heating rate, and the cumulative heat strain invoked during HS. HSP72 and HSP70 gene transcripts were measured in the left ventricle of the heart by means of Western immunoblotting and semiquantitative RT-PCR, respectively. The resultant acclimatory change comprised a higher resting level of the encoded 72-kDa protein (Delta175%, P < 0.0001). After HS, peak HSP72 mRNA level was attained, 40 and 20 min post-HS at 41 and 43 degrees C, respectively, vs. 60 and 40 min in the nonacclimated group. The subsequent HSP synthesis, however, was dependent on the severity of the cumulative heat strain. At the initial phase of heat acclimation, augmented HSP72 transcription unaccompanied by HSP synthesis was observed. It is concluded that upon heat acclimation, the HSP defense pathway is predisposed to a faster response. At the initial phases of heat acclimation, inability to elevate the HSP cytosolic level rules out their direct cytoprotective role.

Short-range linkage relationships, genomic organisation and sequence comparisons of a cluster of five HSP70 genes in Fugu rubripes.

Twelve cosmids containing sequences resembling genes encoding members of the 70-kDa heat-shock protein family, HSP70. have been isolated from Fugu rubripes. They can be broadly divided into three groups of overlapping cosmids. Restriction analysis and sequencing of one set of five cosmids have revealed five intronless Fugu HSP70 genes spanning 42 kb, arranged in a combined head-to-head, tail-to-tail and head-to-tail orientation. The levels of DNA and amino acid identity are very high with respect to one another, and are most similar to HSP70 sequences linked to the major histocompatibility complex (MHC) region in other species. Putative heat-shock consensus elements are identified. Non-HSP70 sequences with homology to known genes have been found physically linked to this Fugu HSP70 cluster: the Drosophila melanogaster SOL gene, the Drosophila melanogaster nemo gene, the Caenorhabditis elegans T17E9.1 gene and the sequence encoding the serine protease domain. The linkage relationships described here so far bear no resemblance to those of HSP70 in other organisms. Convergence of mammalian HSP70 and MHC class I and II loci probably occurred after fish had diverged.

Site-directed Spin Labeling Study of Subunit Interactions in the α-Crystallin Domain of Small Heat-shock Proteins: COMPARISON OF THE OLIGOMER SYMMETRY IN αA-CRYSTALLIN, HSP 27, and HSP 16.3

Site-directed spin labeling was used to investigate quaternary interactions along a conserved sequence in the alpha-crystallin domain of alphaA-crystallin, heat-shock protein 27 (HSP 27), and Mycobacterium tuberculosis heat-shock protein (HSP 16.3). In previous work, it was demonstrated that this sequence in alphaA-crystallin and HSP 27 forms a beta-strand involved in subunit contacts. In this study, the symmetry and geometry of the resulting interface were investigated. For this purpose, the pattern of spin-spin interactions was analyzed, and the number of interacting spins was determined in alphaA-crystallin and HSP 27. The results reveal a 2-fold symmetric interface consisting of two beta-strands interacting near their N termini in an antiparallel fashion. Remarkably, subunit interactions along this interface persist when the alpha-crystallin domains are expressed in isolation. Because this domain in alphaA-crystallin forms dimers and tetramers, it is inferred that interactions along this interface mediate the formation of a basic dimeric unit. In contrast, in HSP 16.3, spin-spin interactions are observed at only one site near the C terminus of the sequence. Furthermore, cysteine substitutions at residues flanking the N terminus resulted in the dissociation of the oligomeric structure. Analysis of the spin-spin interactions and size exclusion chromatography indicates a 3-fold symmetric interface. Taken together, our results demonstrate that subunit interactions in the alpha-crystallin domain of mammalian small heat-shock proteins assemble a basic building block of the oligomeric structure. Sequence divergence in this domain results in variations in the size and symmetry of the quaternary structure between distant members of the small heat-shock protein family.

Vitamin D Receptor Interacts with DnaK/Heat Shock Protein 70: Identification of DnaK Interaction Site on Vitamin D Receptor

Vitamin D receptor (VDR) regulates the expression of vitamin D-dependent genes upon binding to its cognate ligand, 1α,25-dihydroxyvitamin D3(1,25(OH)2D3). This process represents a complex interaction of ligand-bound VDR with nuclear proteins like retinoid X receptor, nuclear accessory factors, and regulatory elements of the target gene. Expression of full-length VDR inEscherichia colirevealed that VDR binds DnaK, a member of heat-shock protein (Hsp) family, with high affinity. By systematic N-terminal truncation of VDR, the interaction site of DnaK on VDR was localized within a 17-amino-acid segment (105–122) representing the “hinge region” between the DNA-binding and hormone-binding domains of VDR. The putative DnaK-binding site was further localized between residues 105 to 109 of VDR by using binding-energy-minimization studies. The interaction of DnaK with VDR did not influence the binding of 1,25(OH)2D3or nuclear accessory factor(s) to VDR. Furthermore, bovine brain Hsp 70, similar to DnaK, interacted with VDR–ligand-binding domain (105–427). These results suggest that DnaK/Hsp 70 may interact with VDR prior to the activation of the latter by 1,25(OH)2D3-binding.

Role of heat shock protein Hsp25 in the response of the orofacial nuclei motor system to physiological stress

Although expression of the small heat shock protein family member Hsp25 has been previously observed in the central nervous system (CNS), both constitutively and upon induction, its function in the CNS remains far from clear. In the present study we have characterized the spatial pattern of expression of Hsp25 in the normal adult mouse brain as well as the changes in expression patterns induced by subjecting mice to experimental hyperthermia or hypoxia. Immunohistochemical analysis revealed a surprisingly restricted pattern of constitutive expression of Hsp25 in the brain, limited to the facial, trigeminal, ambiguus, hypoglossal and vagal motor nuclei of the brainstem. After hyperthermia or hypoxia treatment, significant increases in the levels of Hsp25 were observed in these same areas and also in fibers of the facial and trigeminal nerve tracts. Immunoblot analysis of protein lysates from brainstem also showed the same pattern of induction of Hsp25. Surprisingly, no other area in the brain showed expression of Hsp25, in either control or stressed animals. The highly restricted expression of Hsp25 implies that this protein may have a specific physiological role in the orofacial motor nuclei, which govern precise coordination between muscles of mastication and the pharynx, larynx, and face. Its rapid induction after stress further suggests that Hsp25 may serve as a specific molecular chaperone in the lower cholinergic motor neurons and along their fibers under conditions of stress or injury.

EXPRESSION AND CELLULAR LOCALIZATION OF THE mRNA FOR THE 25-kDa HEAT-SHOCK PROTEIN IN THE MOUSE

The 25-kDa heat-shock protein (Hsp25) is a member of the small heat-shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot andin situhybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin,eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.

The small heat-shock protein, αb-crystallin, has a variable quaternary structure

αB-crystallin is a major structural protein in the lens that is found in a variety of other tissues and is associated with numerous neurological disorders. It is a member of the small heat-shock protein family and possesses chaperone-like properties. Cryo-electron microscopy has been applied to analyze the quaternary structure of human recombinant αB-crystallin, which spontaneously forms roughly spherical multimers 8 to 18 nm in diameter. Class-sum images based on nearly 5000 αB-crystallin particles reveal the presence of a large central cavity, weak regions of density within the protein shell, and an asymmetric quaternary structure. The class-sum images are variable in size and shape, and are suggestive of snapshots of a conformationally flexible assembly. As gel-filtration chromatography reveals a range of molecular masses (650 (±140) kDa) for the assembly, the class-sum images were further classified on the basis of total molecular mass. A reconstruction at ∼4 nm resolution was calculated from the images assigned to 32 subunit (∼645 kDa) assemblies. Comparison of class-sum images with reprojections of the reconstruction indicates that the resolution is limited by the variable nature of the assembly. A three-dimensional variance map indicates significant structural divergence within the protein shell and on the outer surface of the particle. Some of the strong variance may correspond to the flexible, exposed C-terminal residues of the αB-crystallin monomers. The variable quaternary structure of αB-crystallin is consistent with the polydisperse size of the assembly and the previously observed subunit exchange between multimers. Thus, we propose that the monomer packing is variable, and that the quaternary structure of the assembly is not completely defined. A variable αB-crystallin quaternary structure may facilitate binding of target proteins in up to stoichiometric ratios.

Differential HSC70 expression during asexual development of Neurospora crassa.

The constitutive and the heat-shock-induced expression of members of heat-shock protein families changed during vegetative development and conidiation of Neurospora crassa as determined by two-dimensional gel electrophoresis. Western blot and ELISA analyses revealed the highest amounts of the constitutive heat-shock protein 70 (HSC70) in conidiating aerial hyphae and dormant conidia. During conidial germination the amount of HSC70 decreased and subsequently increased during vegetative growth. Stationary mycelia and young aerial hyphae exhibited the lowest HSC70 level. The stationary-phase-dependent decrease in HSC70 was accompanied by a concomitant increase in its nuclear localization, whereas no significant changes in the amount of nuclear HSC70 were found during aerial hyphae development. The cAMP content during aerial hyphae development was inversely correlated with that of HSC70. To examine possible causal relations between HSC70 expression and cAMP content, the adenylate-cyclase-deficient mutant crisp (cr-1) was analysed, which exhibits low concentrations of endogenous cAMP. This mutant, however, showed a lower constitutive HSC70 level, compared to the bdA strain. Treatment of the bd strain and cr-1 mutant with 20 microM 8-bromo-cAMP did not result in significant changes of the constitutive HSC70 level, but in the level of heat-induced HSC/HSP70. In a developmental mutant (acon-2) which is defective in a differentiation step toward conidiation, the expression of HSC70 in aerial hyphae was delayed until the first proconidial chains were observed. It is concluded that the differential expression of HSC/HSP70 does not depend on different nuclear levels of HSC70 or on changes in cAMP concentrations, but rather on developmental genes controlling conidiation.

A 77-kilodalton protein of Cryptococcus neoformans, a member of the heat shock protein 70 family, is a major antigen detected in the sera of mice with pulmonary cryptococcosis.

Heat shock proteins (HSPs) from several pathogenic microbes have been shown to be target molecules of humoral responses as well as cellular immune responses. However, little is known about target molecules in pulmonary cryptococcosis. Western blotting analysis revealed that experimentally induced pulmonary cryptococcosis in (BALB/c x DBA/2)F1 mice was associated with the appearance of serum antibodies to a 77-kDa protein derived from Cryptococcus neoformans as well as to 18-, 22-, 25-, 36-, and 94-kDa proteins. Since the 77-kDa band also reacted with rabbit polyclonal antibodies against 70-kDa HSP (HSP70) family members, the protein was predicted to be a member of the HSP70 family. We also purified HSP70 directly from a C. neoformans cell extract by Mono Q fast protein liquid chromatography and ATP-agarose affinity column chromatography and showed that it was positive in immunoblot analysis using either serum from C. neoformans-infected mice or rabbit anti-HSP70 antibodies. N-terminal amino acid sequencing of this purified protein confirmed that the 77-kDa protein was a member of the HSP70 protein family. A 66-kDa protein, which coincidentally purified with the HSP70 protein and was identified as a member of the HSP60 family by N-terminal amino acid sequencing, was not reactive with sera from C. neoformans-infected mice. Thus, a protein associated with the HSP70 family and derived from C. neoformans was a major target molecule of the humoral response in murine pulmonary cryptococcosis.

The Protein Import System of Mitochondria

The Protein Import System of Mitochondria

Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4–5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.

Analysis of cloned cDNAs differentially expressed in adapting remnant small intestine after partial resection.

After partial resection, the remnant small intestine undergoes an adaptive response. Little is known about the molecular and cellular basis of intestinal adaptation. To identify genes transcriptionally regulated in response to loss of functional bowel surface area, we have isolated cDNAs differentially expressed in the adaptive ileum 48 h after 70% proximal small intestinal resection. A cDNA library constructed from the remnant ileum of rats subjected to resection was screened using subtractive hybridization techniques. Several groups of cDNA clones that were induced during intestinal adaptation were isolated. The first included liver fatty acid binding protein, apolipoprotein A-IV, cellular retinol binding protein II, and ileal lipid binding protein. These all encode proteins involved in the absorption, metabolism, and trafficking of nutrients. A second group included the catalytic subunit of protein phosphatase 1 delta, a 78-kDa glucose-regulated protein (grp 78; a glucose-regulated member of the 70-kDa heat-shock protein family), and several pancreatitis-associated proteins. A third group of induced genes contained novel cDNAs. To better characterize the adaptive response, the temporal, spatial, and cellular patterns of expression of several of these genes were analyzed with the use of immunohistochemical and in situ hybridization techniques. These studies indicate that during early adaptation, genes involved in nutrient trafficking, protein processing, and cell cycle regulation are transcriptionally regulated in the residual small intestine in distinct temporal and regional patterns consistent with a complex multifaceted response to intestinal resection.

Structural analysis of substrate binding by the molecular chaperone DnaK.

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.

During canine viscero-cutaneous leishmaniasis the anti-Hsp70 antibodies are specifically elicited by the parasite protein

A Leishmania infantum cDNA library was screened with sera from dogs with viscero-cutaneous leishmaniasis. Sequence analysis of a positive clone isolated from the library revealed that it coded for the carboxyl-terminal region of a member of the 70-kDa heat-shock protein family. The full-length sequence of the L. infantum hsp70 gene was determined after isolation of genomic clones. This protein shows a high degree of sequence conservation with the homologous protein from other organisms. To test its antigenicity a recombinant Hsp70 protein fused to the maltose-binding protein was produced in Escherichia coli using the expression vector pMAL-cRI. By FAST-ELISA assays it was observed that while the complete recombinant protein was recognized by 100% of the sera, the 20 carboxyl-terminal amino acids of the protein were only recognized by 30% of those sera. Thus, although a B-cell epitope must be present within the carboxyl terminal end of the protein other antigenic determinant(s) must reside out of this region. The analysis of the cross-reactivity with mouse Hsp70 by Western blotting strongly suggests that the anti-Hsp70 antibodies generated by infection with L. infantum are directed at specific determinants of the L. infantum Hsp70. Thus, our results indicate that anti-Hsp70 autoantibodies are not induced during Leishmania infection.

Thermoregulatory and heat-shock protein response deficits in cold-exposed diabetic mice.

Cold-induced expression of heat-shock proteins (HSPs) has been suggested to facilitate thermogenesis in brown adipose tissue (BAT). However, the regulation of this response and the mechanism supporting this facilitation have not been established. Because of the significant role of insulin in maintaining BAT thermogenesis, we employed a transgenic mouse model of diabetes to investigate the regulation and function of HSPs in BAT thermogenesis. These transgenic mice overexpress a calmodulin minigene regulated by the rat insulin II promotor, resulting in severe diabetes characterized by elevated blood glucose and glucagon that coincides with reduced serum and pancreatic insulin. Body temperature (Tb) of diabetic mice dropped significantly faster during a 3-h cold exposure (6 degrees C) than Tb of similarly treated control littermates. Cold exposure resulted in increased levels of constitutive and inducible HSP70 transcripts in control mice, but only constitutive HSP70 mRNA transcripts were induced in diabetic mice. Diabetes did not affect uncoupling protein induction, but cold-induced expression of members of other HSP families was reduced. Correspondingly, heat-shock regulatory factors were not activated in diabetic mice even though these factors were present. Phenylephrine induced HSP70 expression in control and diabetic animals, indicating that alpha-receptor-coupled HSP induction remained intact in BAT of diabetic mice. Insulin replacement restored the Tb response of diabetic mice as well as the HSP response. From these results it is clear that physiological signals that regulate cold-induced activation of BAT also regulate HSP expression in this tissue. This diabetic model provides a novel system in which the HSP response to cold has been selectively knocked out, making it a useful tool for the study of HSP regulation and function in BAT.

Cloning and characterization of the Actinobacillus actinomycetemcomitans gene encoding a heat-shock protein 90 homologue

In a search for clones from a λgtl 1 expression library of Actinobacillus actinomycetemcomitans (Aa) genomic DNA that expressed epitopes from a 70-kDa iron-repressible membrane protein, we inadvertently identified clones that encoded a member of the 90-kDa heat-shock protein (HSP 90) family. The gene appears to encode a homologue of HtpG, as the nucleotide sequence has ∼70% identity with the Escherichia coli ( Ec) and Vibrio fischeri htpG. Growth of an Aa htpG insertion mutant at 42°C was reduced to 50% of the parent strain, similar to an Ec htpG deletion mutant. These data suggest that Aa HtpG performs a function similar to Ec HtpG.

CDNA cloning of a 20-kDa protein (p20) highly homologous to small heat shock proteins: developmental and physiological changes in rat hindlimb muscles

A cDNA clone encoding p20, a novel member of the small heat-shock protein family in mammals, was isolated from a rat soleus cDNA library. The clone contained an insert of 1.3 kb with an open reading frame specifying a polypeptide of 162 amino-acid residues. Southern blot analysis suggested that the p20 gene is a single gene in rat genome. Developmental changes and a sciatic nerve denervation experiment suggested that the expression of p20 in rat hindlimb muscle is related to muscle contraction, and specifically in slow-twitch muscles.

Selective proteolysis: 70-kDa heat-shock protein and ubiquitin-dependent mechanisms?

The proteolysis of the vast majority of cytoplasmic proteins, once inside the lysosome compartment, would be expected to occur rapidly; therefore, the rate-limiting step in the lysosomal degradation of cytoplasmic proteins must be their initial sequestration. Indeed, the initial sequestration serves to remove cytoplasmic proteins from their functional site in the cell. Bulk sequestration of cytoplasm (macro-autophagy), described elsewhere in this volume (Chapter 4), is considered essentially nonselective. Microautophagy has been described by a number of investigators over the years (see Chapter 4) and may provide a mechanism for the selective sequestration of cytoplasmic proteins into the lysosome system. However, controversy surrounds the whole topic of selective lysosomal proteolysis, with some investigators probably of the view that the lysosome system is entirely nonselective. However, recent work from the laboratory of Dice (Dice, 1990) describes a selective mechanism of sequestration of cytosolic proteins into lysosomes for degradation in a process regulated by serum and involving a member of the 70-kDa heat-shock protein family.

Stathmin interaction with a putative kinase and coiled-coil-forming protein domains.

Stathmin is a ubiquitous, cytosolic 19-kDa protein, which is phosphorylated on up to four sites in response to many regulatory signals within cells. Its molecular characterization indicates a functional organization including an N-terminal regulatory domain that bears the phosphorylation sites, linked to a putative alpha-helical binding domain predicted to participate in coiled-coil, protein-protein interactions. We therefore proposed that stathmin may play the role of a relay integrating diverse intracellular regulatory pathways; its action on various target proteins would be a function of its combined phosphorylation state. To search for such target proteins, we used the two-hybrid screen in yeast, with stathmin as a "bait." We isolated and characterized four cDNAs encoding protein domains that interact with stathmin in vivo. One of the corresponding proteins was identified as BiP, a member of the hsp70 heat-shock protein family. Another is a previously unidentified, putative serine/threonine kinase, KIS, which might be regulated by stathmin or, more likely, be part of the kinases controlling its phosphorylation state. Finally, two clones code for subdomains of two proteins, CC1 and CC2, predicted to form alpha-helices participating in coiled-coil interacting structures. Their isolation by interaction screening further supports our model for the regulatory function of stathmin through coiled-coil interactions with diverse downstream targets via its presumed alpha-helical binding domain. The molecular and biological characterization of KIS, CC1, and CC2 proteins will give further insights into the molecular functions and mechanisms of action of stathmin as a relay of integrated intracellular regulatory pathways.