Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.     

Interaction of human HSP22 (HSPB8) with other small heat shock proteins

Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i). HSP22 forms high molecular mass complexes in the heart, (ii). HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii). HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner.     

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear-encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150-fold during a 2-h heat shock. Hybridization-selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5-6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy-terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.     

Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle

Displacement of the contractile protein tropomyosin from actin filament exposes the myosin-binding sites on actin, resulting in actin-myosin interaction and muscle contraction. The objective of the present study was to better understand the interaction of tropomyosin with heat shock protein (HSP)27 in contraction of smooth muscle cells of the colon. We investigated the possibility of a direct protein-protein interaction of tropomyosin with HSP27 and the role of phosphorylated HSP27 in this interaction. Immunoprecipitation studies on rabbit smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that the association of tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)-tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to phosphomimic HSP27 mutant compared with wild-type and nonphosphomimic HSP27. Our data suggest that, in response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin. acetylcholine; fusion proteins; serine     

Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells

One of the monoclonal antibodies raised against mitotic HeLa cells (termed as mH3) recognized a 27-kDa protein and stained microtubules in the mitotic spindles of HeLa cells. Immunoscreening of a HeLa cDNA library revealed that mH3 antigen is a small heat shock protein, HSP27. Immunoprecipitation analysis using mH3 suggested that both alpha- and beta-tubulin are associated with HSP27. Further, sucrose-cushioned ultra centrifugation revealed that HSP27 is co-sedimented with taxol-stabilized microtubules. These results indicate that HSP27 associates with tubulin/microtubules in HeLa cells.     

Association of HSPB2, a member of the small heat shock protein family, with mitochondria.

We previously identified HSPB2, a new member of the small heat shock protein family, expressed in heart and skeletal muscles. In this study, we used a polyclonal anti-HSPB2 antibody and examined the subcellular localization of HSPB2 in differentiated C2C12 cells, KNS-81 cells, and NIH3T3 transfectants expressing human HSPB2. Double staining with anti-HSPB2 and various markers for cytoplasmic structures showed that HSPB2 was present in the cytosol as granules, some of which colocalized with mitochondria. This colocalization was not altered by a colchicine treatment, indicating that it is independent of microtubules. The subcellular fractionation of differentiated C2C12 cells revealed that HSPB2 was mainly detected in the postmitochondrial supernatant, but mild heat treatment enriched the amount of HSPB2 in the mitochondrial fraction. The expression of HSPB2 protected the cells from heat-induced cell death. In addition, Northern blot analysis revealed that expression of HSPB2 mRNA is higher in slow-twitch muscle than in fast-twitch muscle, which correlates with the amounts of mitochondria present in these two types of tissue. Taken together, these results suggest that HSPB2 may not localize in the matrix, but rather associates with the outer membrane components of the mitochondria and thus plays a role in the stress response.     

Diminished phosphorylation of a heat shock protein (HSP 27) in infant acute lymphoblastic leukemia

We have previously reported lack of expression of a polypeptide designated L3 in infant acute lymphoblastic leukemia (ALL). Expression of L3 occurred predominantly in older children with pre-B ALL. We have recently reported the expression during B cell ontogeny of two other polypeptides, designated L2 and L4 with a similar Mr as L3, which were identified as phosphorylated and non-phosphorylated forms respectively of the low Mr heat shock protein. hsp27. In this study we have characterized L3 and identified it as another phosphorylated form of hsp27. The two phosphorylated forms appear to be differentially expressed in acute leukemia. L3 levels in infants who expressed hsp27 isoforms L2 and L4 were significantly diminished compared to levels in older children with an equivalent amount of hsp27. We conclude that leukemic cells in infant ALL exhibit a unique pattern of phosphorylation of hsp27 expressed at a pre-B cell stage of differentiation.     

In vivo modifications of the maize mitochondrial small heat stress protein, HSP22

A maize (Zea mays L.) small heat shock protein (HSP), HSP22, was previously shown to accumulate to high levels in mitochondria during heat stress. Here we have purified native HSP22 and resolved the protein into three peaks using reverse phase high performance liquid chromatography. Mass spectrometry (MS) of the first two peaks revealed the presence of two HSP22 forms in each peak which differed in mass by 80 daltons (Da), indicative of a monophosphorylation. Phosphorylation of HSP22 by [gamma-(32)P]ATP was also observed in mitochondria labeled in vitro, but not when purified native HSP22 was similarly used, demonstrating that HSP22 does not autophosphorylate, implicating a kinase involvement in vivo. Collisionally induced dissociation tandem MS (CID MS/MS) identified Ser(59) as the phosphorylated residue. We have also observed forms of HSP22 that result from alternative intron splicing. The two HSP22 proteins in the first peak were approximately 57 Da larger than the two HSP22 proteins in the second peak. MS analysis revealed that the +57-Da forms have an additional Gly residue directly N-terminal of the expected Asp(84), which had been converted to an Asn residue. These results are the first demonstrations of phosphorylation and alternative intron splicing of a plant small HSP.     

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with K_D ~ 1 μM. Proteins co-localize in the soluble fraction of the cell at 30–37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpA_His6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30–37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.     

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.     

ERp27, a new non-catalytic endoplasmic reticulum-located human protein disulfide isomerase family member, interacts with ERp57

Protein folding and quality control in the endoplasmic reticulum are critical processes for which our current understanding is far from complete. Here we describe the functional characterization of a new human 27.7-kDa protein (ERp27). We show that ERp27 is a two-domain protein located in the endoplasmic reticulum that is homologous to the non-catalytic b and b' domains of protein disulfide isomerase. ERp27 was shown to bind Delta-somatostatin, the standard test peptide for protein disulfide isomerase-substrate binding, and this ability was localized to the second domain of ERp27. An alignment of human ERp27 and human protein disulfide isomerase allowed for the putative identification of the peptide binding site of ERp27 indicating conservation of the location of the primary substrate binding site within the protein disulfide isomerase family. NMR studies revealed a significant conformational change in the b'-like domain of ERp27 upon substrate binding, which was not just localized to the substrate binding site. In addition, we report that ERp27 is bound by ERp57 both in vitro and in vivo by a similar mechanism by which ERp57 binds calreticulin.     

BH-protocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain

Using a yeast two-hybrid system, we isolated eight cDNA clones which interacted with BH-protocadherin-c (BH-Pcdh-c) from the human brain cDNA library. One clone encoded protein phosphatase type I isoform alpha (PP1alpha) and another two PP1alpha2. PP1alpha was co-immunoprecipitated from the extract of a gastric adenocarcinoma cell line MKN-28 with anti-BH-Pcdh-c antibody. PP1alpha activity towards glycogen phosphorylase was inhibited by the intracellular domain of BH-Pcdh-c. Inhibition of the phosphatase required more than the minimal domain of BH-Pcdh-c which could associate with PP1alpha. In situ hybridization revealed that BH-Pcdh-c mRNA was predominantly expressed in cerebral cortex neurons in the adult mouse brain.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

Effects of the overexpression of the small heat shock protein,HSP27, on the sensitivity of human fibroblast cells exposed to oxidative stress

The role of the human small heat shock protein (HSP27) in oxidative stress was examined using stable transformants of an immortalized human fibroblast cell line (KMST-6) isolated by transfection of HSP27 expression vectors. Several stable transformants that expressed high or low levels of HSP27 protein were obtained. Clones expressing high levels of HSP27 were more sensitive to growth inhibition by a low dose of hydrogen peroxide (0.1 mM) than those expressing low levels. Clones expressing high levels of HSP27 did not acquire obvious resistance to hyperthermy and cytotoxic agents, except for one (#13), in which resistance to cytotoxic agents was increased. The level of phosphorylated HSP27 in clones expressing high levels of this protein increased at 30 min and was sustained even 4 hours after exposing the cells to 0.1 mM of hydrogen peroxide. On the other hand, the levels in clones expressing low levels of HSP27 were reduced within 4 hours after exposure to hydrogen peroxide. Furthermore, overexpression of nonphosphorylatable mutant HSP27 did not affect sensitivity to oxidative stress. These results suggested that constitutively high expression of HSP27 in KMST-6 cells make them susceptible to oxidative stress resulting in growth arrest, and this mechanism could involve the phosphorylation of HSP27. © 1995 Wiley-Liss, Inc.     

A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock.

A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus. In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation. Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule. The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli.