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HSP22, a New Member of the Small Heat Shock Protein Superfamily, Interacts with Mimic of Phosphorylated HSP27 (3DHSP27)

Open AccessPublished:July 20, 2001DOI:https://doi.org/10.1074/jbc.M103001200
      Most of the members of the superfamily of mammalian small heat shock or stress proteins are abundant in muscles where they play a role in muscle function and maintenance of muscle integrity. One member of this protein superfamily, human HSP27, is rapidly phosphorylated on three serine residues (Ser15, Ser78, and Ser82) during cellular response to a number of extracellular factors. To understand better the role of HSP27, we performed a yeast two-hybrid screen of a human heart cDNA library for HSP27-interacting proteins. By using the triple aspartate mutant, a mimic of phosphorylated HSP27, as “bait” construct, a protein with a molecular mass of 21.6 kDa was identified as an HSP27-binding protein. Sequence analysis revealed that this new protein shares an overall sequence identity of 33% with human HSP27. This protein also contains the α-crystallin domain in its C-terminal half, a hallmark of the superfamily of small stress proteins. Thus, the new protein itself is a member of this protein superfamily, and consequently we designated it HSP22. According to the two-hybrid data, HSP22 interacts preferentially with the triple aspartate form of HSP27 as compared with wild-type HSP27. HSP22 is expressed predominantly in muscles. In vitro, HSP22 is phosphorylated by protein kinase C (at residues Ser14 and Thr63) and by p44 mitogen-activated protein kinase (at residues Ser27 and Thr87) but not by MAPKAPK-2.
      sHSP
      mammalian small heat shock protein
      HSP20
      HSP22, HSP27 (also referred to as HSP25), mammalian small heat shock proteins 20, 22, 27, respectively
      hHSP22
      rHSP22, mHSP22, human, rat, mouse HSP22, respectively
      wthHSP27
      3DhHSP27, wild-type and mutant form of hHSP27
      rHSP27
      rat HSP27
      RT-PCR
      reverse transcriptase polymerase chain reaction
      PAGE
      polyacrylamide gel electrophoresis
      IEF
      isoelectric focusing
      PKC
      protein kinase C
      PKA
      protein kinase A
      PKG
      protein kinase G
      MAPK
      mitogen-activated protein kinase
      MAPKAPK-2
      mitogen-activated protein kinase-activated protein kinase-2
      CK-2
      casein kinase-2
      PVDF
      polyvinylidene difluoride
      MS/MS
      tandem mass spectrometry
      bp
      base pair
      Q-TOF
      quadruple-time-of-flight
      The superfamily of mammalian small heat shock or stress proteins (sHSP)1 in humans consists of the known members HSP27, αB-crystallin, αA-crystallin, HSP20, HSPB2, HSPB3, and cvHSP (
      • Kato K.
      • Goto S.
      • Inaguma Y.
      • Hasegawa K.
      • Morishita R.
      • Asano T.
      ,
      • de Jong W.W.
      • Caspers G.-J.
      • Leunissen J.A.M.
      ,
      • Boelens W.C.
      • Van Boekel M.A.
      • de Jong W.W.
      ,
      • Suzuki A.
      • Sugiyama Y.
      • Hayashi Y.
      • Nyu-i N.
      • Yoshida M.
      • Nonaka I.
      • Ishiura S.-I.
      • Arahata K.
      • Ohno S.
      ,
      • Krief S.
      • Faivre J.-F.
      • Robert P.
      • LeDouarin B.
      • Brument-Larignon N.
      • Lefrere I.
      • Bouzyk M.M.
      • Anderson K.M.
      • Greller L.D.
      • Tobin F.L.
      • Souchet M.
      • Bril A.
      ). Throughout the animal, plant, and microbiotic kingdoms, members of this protein superfamily share the so-called α-crystallin domain in their C-terminal part, whereas other parts of the sequence (N-terminal halves and extreme C-terminal tails) are more variable. Some of the sHSPs are known to be phosphoproteins. αB-crystallin is phosphorylated at three sites by the protein kinases p44/42 MAPK (Erk1/2), MAPKAPK-2, and probably by PKA (
      • Kato K.
      • Ito H.
      • Kamei K.
      • Inaguma Y.
      • Iwamoto I.
      • Saga S.
      ,
      • Voorter C.E.
      • de Haard-Hoekman W.A.
      • Roersma E.S.
      • Meyer H.E.
      • Bloemendal H.
      • de Jong W.W.
      ), whereas HSP20 possesses at least one PKG/PKA phosphorylation site (
      • Beall A.
      • Bagwell D.
      • Woodrum D.
      • Stoming T.A.
      • Kato K.
      • Suzuki A.
      • Rasmussen H.
      • Brophy C.M.
      ). The best studied is HSP27, which is phosphorylated at two (mouse) or three (human) phosphorylation sites by MAPKAPK-2 (
      • Gaestel M.
      • Schröder W.
      • Benndorf R.
      • Lippmann C.
      • Buchner K.
      • Hucho F.
      • Erdmann V.A.
      • Bielka H.
      ,
      • Landry J.
      • Lambert H.
      • Zhou M.
      • Lavoie J.N.
      • Hickey E.
      • Weber L.A.
      • Anderson C.W.
      ,
      • Stokoe D.
      • Engel K.
      • Campbell D.G.
      • Cohen P.
      • Gaestel M.
      ). Phosphorylation of HSP27 is an early and prominent event in many cells when stimulated by a variety of mitogenic and stress factors. For example, contraction of smooth muscle cells in response to bombesin or endothelin-1 involves phosphorylation of HSP27 (
      • Bitar K.N.
      • Kaminski M.S.
      • Hailat N.
      • Cease K.B.
      • Strahler J.R.
      ,
      • Yamboliev I.A.
      • Hedges J.C.
      • Mutnick J.L.-M.
      • Adam L.P.
      • Gerthofer W.T.
      ). Similarly, Sertoli cells respond to germ cells (
      • Pittenger G.L.
      • Gilmont R.R.
      • Welsh W.J.
      ) and pancreatic acinar cells to cholecystokinin (
      • Schäfer C.
      • Clapp P.
      • Welsh M.J.
      • Benndorf R.
      • Williams J.A.
      ) by rapid phosphorylation of HSP27. Another characteristic of sHSPs is their ability to form supramolecular structures (complexes). The basic structural units of sHSP complexes are dimers that are formed by interaction of segments of the α-crystallin domain (
      • Liu C.
      • Welsh M.J.
      ). The formation of structures of higher orders, such as tetramers, octomers, etc., appears to require additional sites of interaction that have been located to the N-terminal region of the molecules (
      • Lambert H.
      • Charette S.J.
      • Bernier A.F.
      • Guimond A.
      • Landry J.
      ). Besides homo-oligomeric complexes, sHSPs can also form hetero-oligomeric complexes if different sHSP species are present (
      • Liu C.
      • Welsh M.J.
      ,
      • Bova M.P.
      • Mchaourab H.S.
      • Han Y.
      • Fung B.K.-K.
      ,
      • Sugiyama Y.
      • Suzuki A.
      • Kishikawa M.
      • Akutsu R.
      • Hirose T.
      • Waye M.M.Y.
      • Tsui S.K.W.
      • Yoshida S.
      • Ohno S.
      ). sHSP complexes have a dynamic structure with subunits exchanging rapidly between complexes as has been determined by fluorescence resonance energy transfer measurements (
      • Bova M.P.
      • Mchaourab H.S.
      • Han Y.
      • Fung B.K.-K.
      ). The molecular mass of cellular sHSP complexes varies over a wide range (50–1000 kDa), with complexes of increasing size being predominant under stress conditions. Complex formation is believed to be a key feature in regulating the activity of sHSPs in vivo, although details are not understood (
      • Preville X.
      • Schultz H.
      • Knauf U.
      • Gaestel M.
      • Arrigo A.-P.
      ). Also, the reported in vitro activities of HSP27, chaperoning and inhibition of actin polymerization, depend positively or negatively, respectively, on complex formation, which itself appears to depend, to a certain extent, on the degree of phosphorylation (
      • Rogalla T.
      • Ehrnsperger M.
      • Preville X.
      • Kotlyarov Z.
      • Lutsch G.
      • Ducasse C.
      • Paul C.
      • Wieske M.
      • Arrigo A.-P.
      • Buchner J.
      • Gaestel M.
      ,
      • Leroux M.R.
      • Melki R.
      • Gordon B.
      • Batelier G.
      • Candido E.P.
      ,
      • Benndorf R.
      • Hayess K.
      • Ryazantsev S.
      • Wieske M.
      • Behlke J.
      • Lutsch G.
      ,
      • Kato K.
      • Hasegawa K.
      • Goto S.
      • Inaguma Y.
      ).
      Besides interaction with themselves, sHSPs can also interact with other proteins. For example, HSP27 binds PASS1 in Sertoli cells (
      • Liu C.
      • Gilmont R.R.
      • Benndorf R.
      • Welsh M.J.
      ), protein kinase B in COS-7 cells (
      • Konishi H.
      • Matsuzaki H.
      • Tanaka M.
      • Takemura Y.
      • Kuroda S.
      • Ono Y.
      • Kikkawa U.
      ), mammalian transglutaminase (platelet factor XIII) in platelets (
      • Zhu Y.
      • Tassi L.
      • Lane W.
      • Mendelsohn M.E.
      ), and actin in vitro (
      • Miron T.
      • Vancompernolle K.
      • Vandekerckhove J.
      • Wilchek M.
      • Geiger B.
      ). HSPB2 binds myotonic dystrophy protein kinase (
      • Suzuki A.
      • Sugiyama Y.
      • Hayashi Y.
      • Nyu-i N.
      • Yoshida M.
      • Nonaka I.
      • Ishiura S.-I.
      • Arahata K.
      • Ohno S.
      ), and αB-crystallin binds vimentin, desmin, and actin (
      • Wang K.
      • Spector A.
      ,
      • Nicoll I.D.
      • Quinlan R.A.
      ,
      • Perng M.D.
      • Muchowski P.J.
      • van den IJssel P.
      • Wu G.J.S.
      • Hutcheson A.M.
      • Clark J.I.
      • Quinlan R.A.
      ). It is not known which factors control the interaction of sHSPs with themselves or with other proteins.
      In recent years, evidence has been obtained for the crucial role of some of the sHSPs in muscle function. A point mutation in the αB-crystallin gene causes a severe desmin-related cardiomyopathy in humans (
      • Vicart P.
      • Caron A.
      • Guicheney P.
      • Li Z.
      • Prevost M.-C.
      • Faure A.
      • Chateau D.
      • Chapon F.
      • Tome F.
      • Dupret J.-M.
      • Paulin D.
      • Fardeau M.
      ), and HSPB2 binds and activates the myotonic dystrophy protein kinase, an enzyme that when absent results in myotonic dystrophy (
      • Suzuki A.
      • Sugiyama Y.
      • Hayashi Y.
      • Nyu-i N.
      • Yoshida M.
      • Nonaka I.
      • Ishiura S.-I.
      • Arahata K.
      • Ohno S.
      ). Whereas in most cell types HSP27 and αB-crystallin have a diffuse cytosolic location, the situation in striated muscles is different. Both proteins have been found in specific locations such as the I-band (
      • Lutsch G.
      • Vetter R.
      • Offhauss U.
      • Wieske M.
      • Gröne H.-J.
      • Klemenz R.
      • Schimke I.
      • Stahl J.
      • Benndorf R.
      ) and Z-disc (
      • Golenhofen N.
      • Htun P.
      • Ness W.
      • Koob R.
      • Schaper W.
      • Drenckhahn D.
      ) of myocytes, and most importantly, they quickly relocate between the I-band, Z-disc, and the cytosol upon disease-related, ischemic, or thermal stress (
      • Lutsch G.
      • Vetter R.
      • Offhauss U.
      • Wieske M.
      • Gröne H.-J.
      • Klemenz R.
      • Schimke I.
      • Stahl J.
      • Benndorf R.
      ,
      • Golenhofen N.
      • Htun P.
      • Ness W.
      • Koob R.
      • Schaper W.
      • Drenckhahn D.
      ,
      • van de Klundert F.A.J.
      • Gijsen M.L.J.
      • van den IJssel P.R.L.A.
      • Snoeckx L.H.E.H.
      • de Jong W.W.
      ). The highly specific location of sHSPs in myocytes and the stress-related relocation of sHSPs led us to hypothesize that HSP27 interacts with other proteins in muscle. It is also hypothesized that the observed relocation of HSP27 involves a regulatory mechanism able to respond to physiological signals.
      In an effort to identify proteins that interact with HSP27 in muscle cells, we screened a human heart cDNA library by the yeast two-hybrid method. In order to obtain data related to the regulation of the binding preference of HSP27 in dependence on its phosphorylation, we used as “bait” proteins both human wild-type HSP27 (wthHSP27) and a mimic of phosphorylated human HSP27 in which the three serines phosphorylated by MAPKAPK-2 (Ser15, Ser78, and Ser82) had been substituted by aspartate (3DhHSP27). Here we report the identification, molecular cloning, and characterization of a novel sHSP we designated HSP22 and that is expressed in striated and smooth muscle tissues. This protein binds preferentially to 3DhHSP27 rather than to wthHSP27. Thus, HSP22 matches the requirements of the initial hypothesis. It is a new binding partner of HSP27 occurring in muscles, and the HSP27-HSP22 interaction is regulated by phosphorylation of HSP27.

      DISCUSSION

      A number of observations suggest that sHSPs, which may constitute 1% or more of total muscular protein (
      • de Jong W.W.
      • Caspers G.-J.
      • Leunissen J.A.M.
      ), play a major role in function and differentiation of muscles and in the maintenance of their integrity under stress conditions. Striking arguments for the involvement of sHSPs in normal muscle function comes from genetic diseases. Autosomal dominant desmin-related cardiomyopathy (
      • Vicart P.
      • Caron A.
      • Guicheney P.
      • Li Z.
      • Prevost M.-C.
      • Faure A.
      • Chateau D.
      • Chapon F.
      • Tome F.
      • Dupret J.-M.
      • Paulin D.
      • Fardeau M.
      ) is caused by the missense mutation R120G in the αB-crystallin gene resulting in an abnormal organization of the intermediate filament protein desmin, a known interacting partner of αB-crystallin. Myotonic dystrophy is caused by a genetic defect in which GTC repeats in the 3′-untranslated region of the gene coding for the myotonic dystrophy protein kinase are unstably transmitted resulting in a lack of this enzyme. However, the complex pattern of symptoms is not simply a matter of lack of this enzyme (
      • Jansen G.
      • Groenen P.J.
      • Bachner D.
      • Jap P.H.
      • Coerwinkel M.
      • Oerlemans F.
      • van den Broek W.
      • Gohlsch B.
      • Pette D.
      • Plomp J.J.
      • Molenaar P.C.
      • Nederhoff M.G.
      • van Echteld C.J.
      • Dekker M.
      • Berns A.
      • Hameister H.
      • Wieringa B.
      ), and the activation and protection of this protein kinase by HSPB2 (
      • Suzuki A.
      • Sugiyama Y.
      • Hayashi Y.
      • Nyu-i N.
      • Yoshida M.
      • Nonaka I.
      • Ishiura S.-I.
      • Arahata K.
      • Ohno S.
      ) is an indication that both proteins are involved in protection of muscles under stress conditions. Also, the differentiation of multipotent embryonic carcinoma cells P19 into functional cardiomyocytes requires the p38/HSP27 pathway (
      • Davidson S.M.
      • Morange M.
      ). Both, HSP27 and αB-crystallin were shown to protect cardiomyocytes from adverse anoxic conditions, a model system for pathological ischemia (
      • Martin J.
      • Mestril R.
      • Hilal-Dandan R.
      • Brunton L.L.
      • Dillmann W.
      ).
      Studies of the intracellular localization of sHSPs in myocytes have also led to interesting observations. In early studies αB-crystallin was found to localize in the Z-disc in adult cardiomyocytes (
      • Longioni S.
      • Lattonen S.
      • Bullock G.
      • Chiesi M.
      ), while later both HSP27 and αB-crystallin were found to localize in the I-band (
      • Lutsch G.
      • Vetter R.
      • Offhauss U.
      • Wieske M.
      • Gröne H.-J.
      • Klemenz R.
      • Schimke I.
      • Stahl J.
      • Benndorf R.
      ) and the cytosol (
      • Yoshida K.
      • Aki T.
      • Harada K.
      • Shama K.M.
      • Kamoda Y.
      • Suzuki A.
      • Ohno S.
      ,
      • Sakamoto K.
      • Urushidani T.
      • Nagao T.
      ). This difference in observations is probably resolved by the fact that sHSPs can re-localize quickly, depending on the physiological conditions. Pathological stress was reported to result in a depletion of HSP27 and αB-crystallin from the sarcomeres into the cytosol (
      • Lutsch G.
      • Vetter R.
      • Offhauss U.
      • Wieske M.
      • Gröne H.-J.
      • Klemenz R.
      • Schimke I.
      • Stahl J.
      • Benndorf R.
      ), whereas ischemia resulted in a relocation of αB-crystallin in the Z-disc and, with increasing severity, also in the I-band (
      • Golenhofen N.
      • Htun P.
      • Ness W.
      • Koob R.
      • Schaper W.
      • Drenckhahn D.
      ). In other experimental systems, ischemia and preconditioning caused a shift of HSP27, HSPB2, and αB-crystallin from the cytosol to the sarcomeres (
      • Yoshida K.
      • Aki T.
      • Harada K.
      • Shama K.M.
      • Kamoda Y.
      • Suzuki A.
      • Ohno S.
      ,
      • Sakamoto K.
      • Urushidani T.
      • Nagao T.
      ). In neonatal cardiomyocytes, in which sHSPs have a basic cytosolic location, thermal stress shifted HSP27 and αB-crystallin into the myofibrils (
      • van de Klundert F.A.J.
      • Gijsen M.L.J.
      • van den IJssel P.R.L.A.
      • Snoeckx L.H.E.H.
      • de Jong W.W.
      ). Recently, phosphorylation and relocation of sHSPs were suggested to play a role in physiological phenomena such as ischemic preconditioning (
      • Eaton P.
      • Awad W.I.
      • Miller J.I.A.
      • Hearse D.J.
      • Shattock M.J.
      ).
      These facts strongly suggest an involvement of sHSPs in muscle function and led us to hypothesize that sHSPs must interact with other proteins in muscles. Additionally, there must be a regulatory mechanism, such as phosphorylation of HSP27, that would change the binding preference of these proteins and that could serve to explain the stress-related relocation. To test this hypothesis, we used the two-hybrid method to screen a human heart cDNA library for proteins interacting with hHSP27, using as bait both wild-type hHSP27 and a mutant that mimics phosphorylated hHSP27. Of the 17 positive yeast clones expressing HSP27-binding proteins, 16 were determined to be sHSPs (hHSP27 itself, hαB-crystallin, hHSP20, and hHSP22). This was an expected result since the heart contains high amounts of these proteins that all contain the α-crystallin domain as the identified interacting site. The abundance of αB-crystallin (3–4 µg/mg) and HSP20 (2 µg/mg) in heart is ∼7.5 and 4.5 times, respectively, higher than HSP27 itself (0.2–0.7 µg/mg). This would explain, together with the ability of these sHSPs to form heterodimers, the high incidence with which these proteins were identified in the screening (
      • Kato K.
      • Goto S.
      • Inaguma Y.
      • Hasegawa K.
      • Morishita R.
      • Asano T.
      ,
      • Lutsch G.
      • Vetter R.
      • Offhauss U.
      • Wieske M.
      • Gröne H.-J.
      • Klemenz R.
      • Schimke I.
      • Stahl J.
      • Benndorf R.
      ,
      • Kato K.
      • Shinohara H.
      • Kurobe N.
      • Inaguma Y.
      • Shimizu K.
      • Ohshima K.
      ,
      • Kato K.
      • Shinohara H.
      • Goto S.
      • Inaguma Y.
      • Morishita R.
      • Asano T.
      ,
      • Inaguma Y.
      • Hasegawa K.
      • Goto S.
      • Ito H.
      • Kato K.
      ). Similarly, the known tendency of HSP27 to form homodimers (
      • Liu C.
      • Welsh M.J.
      ,
      • Zavialov A.
      • Benndorf R.
      • Ehrsperger M.
      • Zav'yalov V.
      • Dudich I.
      • Zav'yalova G.
      • Buchner J.
      • Gaestel M.
      ) explains the high incidence with which HSP27 itself was identified. More difficult to explain, however, is the different frequencies with which both bait constructs were found to bind to the other sHSPs in this screening. αB-Crystallin was identified 5 timesversus 2 times, HSP20 2 times versus 0 times, and HSP27 0 times versus 6 times, using as baitswtHSP27 and 3DHSP27, respectively. These data may indicate that there is a general change in the binding preference of HSP27 upon phosphorylation. The possible consequences of this include alterations in the composition of sHSP complexes. This has been observed in several experimental systems (
      • Rogalla T.
      • Ehrnsperger M.
      • Preville X.
      • Kotlyarov Z.
      • Lutsch G.
      • Ducasse C.
      • Paul C.
      • Wieske M.
      • Arrigo A.-P.
      • Buchner J.
      • Gaestel M.
      ,
      • Kato K.
      • Hasegawa K.
      • Goto S.
      • Inaguma Y.
      ,
      • Lavoie J.N.
      • Lambert H.
      • Hickey E.
      • Weber L.A.
      • Landry J.
      ,
      • Mehlen P.
      • Arrigo A.-P.
      ). The preferential binding of 3DhHSP27 (as opposed towthHSP27) to hHSP22 strongly suggests such a change in binding preference upon HSP27 phosphorylation thus providing an argument for this view. In fact, these findings shed new light on the molecular function of HSP27 phosphorylation that may have consequences for the activity of sHSPs in a number of biological systems. For example, this shift in the binding preference among different sHSPs may well be the basis for the relocation of HSP27 and αB-crystallin observed in muscle cells and may be involved in responses such as ischemic preconditioning of hearts (
      • Eaton P.
      • Awad W.I.
      • Miller J.I.A.
      • Hearse D.J.
      • Shattock M.J.
      ).
      hHSP22 is most closely related to hHSP27 among the known human members of this protein superfamily according to a parsimony analysis (not shown). Similarity is highest in the α-crystallin domain with the common motifs VKTKDK, GKHEE, VDP, SLSPEG, and EAP (cf. Fig.1 D), and it is less pronounced in the N-terminal part with only a few common motifs (DPFRD and SLR) and in the C-terminal tail. Thus, HSP22 fits into the similarity pattern characteristic for this protein superfamily (
      • de Jong W.W.
      • Caspers G.-J.
      • Leunissen J.A.M.
      ). The three MAPKAPK-2 phosphorylation sites of hHSP27 (Ser15, Ser78, and Ser82) are not conserved in the hHSP22 sequence, and the only possible MAPKAPK-2 site at Ser58 in the sequence LPRLSS(cf. Fig. 1 E) is not functional (Fig.5 A), probably due to steric hindrance. In transfected COS-7 cells, hHSP22 exists in three isoforms (pI ∼4.9, 4.6, and 4.3). The pI of the most basic isoform (4.9) is close to the predicted pI of 5.0. The more acidic isoforms may originate from modifications such as phosphorylation; however, to date the nature of these modifications is not known. Like other sHSPs studied, hHSP22 has a slightly higher apparent molecular mass than predicted (25 versus 22 kDa), and it has the ability to form homodimers (cf. Fig.4 B). Whether HSP22 also can form heterodimers with sHSPs other than 3DHSP27 and larger oligomers (complexes) is not known so far but seems reasonable to assume. On Northern blots, hHSP22 mRNA was detected in a single band of ∼1.8 kilobase pairs which is slightly larger than the size predicted from the sequence shown in Fig. 1 A, probably due to the addition of a poly(A) tail.
      While this study was in progress, sequences identical with hHSP22 were submitted to GenBankTM (GenBankTMaccession numbers AAF09481, NP055180, and AAD55359), and one report was published describing this sequence (
      • Smith C.C., Yu, Y.X.
      • Kulka M.
      • Aurelian L.
      ). In that study, the α-crystallin domain of hHSP22 was not recognized, as well as the similarity of this protein with the other sHSPs. hHSP22 (designated H11) was identified as a mammalian protein with weak similarity to ICP10, a herpes simplex virus type 2 ribonucleotide reductase. This protein was suggested to have a weak autophosphorylation activity similar to what has been reported earlier for αB-crystallin (
      • Kantorow M.
      • Piatigorsky J.
      ). The basis for the residual protein kinase activity of these sHSPs is the presence of a few (∼5) catalytic motifs in the α-crystallin domain, whereas a fully active protein kinase usually has 12 motifs. Despite this residual activity, protein phosphorylation or autophosphorylation is probably not the primary function of sHSPs including HSP22.
      The expression of HSP22 varies in different human tissues, although the prevailing expression in all three types of muscles is evident (cf. Fig. 3). Hence, the expression pattern of HSP22 is similar to that of most of the other mammalian sHSPs that are, with the exception of αA-crystallin, abundant in muscles (
      • de Jong W.W.
      • Caspers G.-J.
      • Leunissen J.A.M.
      ,
      • Krief S.
      • Faivre J.-F.
      • Robert P.
      • LeDouarin B.
      • Brument-Larignon N.
      • Lefrere I.
      • Bouzyk M.M.
      • Anderson K.M.
      • Greller L.D.
      • Tobin F.L.
      • Souchet M.
      • Bril A.
      ,
      • Sugiyama Y.
      • Suzuki A.
      • Kishikawa M.
      • Akutsu R.
      • Hirose T.
      • Waye M.M.Y.
      • Tsui S.K.W.
      • Yoshida S.
      • Ohno S.
      ). In striated muscles, sHSPs have been found to form two types of complexes, with type I complex consisting of HSP27, HSP20, and αB-crystallin and type II complex consisting of HSPB2 and HSPB3 (
      • Sugiyama Y.
      • Suzuki A.
      • Kishikawa M.
      • Akutsu R.
      • Hirose T.
      • Waye M.M.Y.
      • Tsui S.K.W.
      • Yoshida S.
      • Ohno S.
      ). In accordance with that, the type I sHSPs, HSP27, HSP20, and αB-crystallin, have been identified as binding partners for HSP27 in the two-hybrid screening in this study. Whether HSP22 is also a part of the type I complex is not known at present; it may also exist in a separate form, e.g.as monomer, dimer, or as a complex. The observed tendency of type I complexes to disassemble upon phosphorylation of HSP27 may be related to the fact that HSP27 shifts its binding preference toward HSP22. In general, sHSP complexes are highly dynamic structures that quickly exchange subunits (
      • Bova M.P.
      • Mchaourab H.S.
      • Han Y.
      • Fung B.K.-K.
      ). So far, no data on the dynamics or structure of complexes involving HSP22 are available.
      There are some indications that hHSP22 is a phosphoprotein. In vivo, using transfected 293T cells, hHSP22 could be labeled with radioactive phosphorous, and the hydrolysis of this labeled hHSP22 revealed phosphoserine and phosphothreonine as phosphoamino acids (
      • Smith C.C., Yu, Y.X.
      • Kulka M.
      • Aurelian L.
      ). This fits with the in vitro data obtained in the present study with serine and threonine residues identified as being phosphorylated by PKC and p44 MAPK (Erk1) (Figs. 1 E and5).
      PKC is a rather unspecific protein kinase requiring basic amino acid residues near the phosphoacceptor group. PKC uses the consensus motif of the general structure (R/K)X 0–2(S/ T)X 0–2(R/K) or its parts (S/T)X 0–2(R/K) and (R/K)X 0–2(S/T) (with up to two amino acid residues between the basic residues and the phosphoacceptor group) (
      • Kennelly P.J.
      • Krebs E.G.
      ). Among the 16 potential phosphorylation sites of hHSP22 for PKC fitting into this general motif, two sites (Ser14-Arg,Thr63-Leu-Arg) were found to be phosphorylated. Both sites fit into the consensus motif and are conserved among the three HSP22 sequences studied (cf. Fig.1 D).
      Proline-directed MAPKs generally use the consensus motif PX(S/T)P or frequently its part (S/T)P (
      • Hawkins J.
      • Zheng S.
      • Frantz B.
      • LoGrasso P.
      ,
      • Davis R.J.
      ,
      • Mukhopadhyay N.K.
      • Price D.J.
      • Kyriakis J.M.
      • Pelech S.
      • Sanghera J.
      • Avruch J.
      ). Among the three potential phosphorylation sites of hHSP22 with the minimal motif (S/T)P, one site (Thr87-Arg) was found to be phosphorylated by p44 MAPK (Erk1) in vitro. However, this site is not conserved among the HSP22 sequences studied (Asn in rat and Ser in mouse) in otherwise highly conserved sequences (cf. Fig.1 D). The significance of this is not known. The other identified phosphorylation site, Ser27, is positioned in the sequence Pro-Leu-Ser27-Ser-Arg. While Pro-Leu-Ser27 would be part of the general motif PX(S/T)P, this minimal sequence is only occasionally used by proline-directed protein kinases. For example, the maturation promoting factor/cdc2 protein kinase has a modest preference for proline at position −2 using epidermal growth factor receptor-derived peptides as substrates (
      • Mukhopadhyay N.K.
      • Price D.J.
      • Kyriakis J.M.
      • Pelech S.
      • Sanghera J.
      • Avruch J.
      ). Furthermore, a basic residue at position +2 (as the arginine in Pro-Leu-Ser27-Ser-Arg) has a strongly positive effect on proline-directed protein kinases (
      • Mukhopadhyay N.K.
      • Price D.J.
      • Kyriakis J.M.
      • Pelech S.
      • Sanghera J.
      • Avruch J.
      ). Thus, the sequence around Ser27 meets the requirements for a potential phosphorylation site used by proline-directed protein kinases, although this type of motif deviates from the most frequently used motifs. Among the two potential phosphorylation sites for proline-directed protein kinases fitting into the motif PX(S/T)X(R/K), one site was found to be phosphorylated. Ser27 is conserved among the HSP22 sequences studied.
      In future experiments, the in vivo relevance of these phosphorylation sites as well as the involved signal transduction cascades will be elucidated. All four HSP22 phosphorylation sites identified in this study are positioned in the N-terminal part of HSP22 as is the case with the phosphorylation sites of the other muscle-relevant sHSPs. According to the current state of knowledge, each sHSP seems to be phosphorylated by a unique set of protein kinases as follows: HSP27 by MAPKAPK-2 (
      • Gaestel M.
      • Schröder W.
      • Benndorf R.
      • Lippmann C.
      • Buchner K.
      • Hucho F.
      • Erdmann V.A.
      • Bielka H.
      ,
      • Landry J.
      • Lambert H.
      • Zhou M.
      • Lavoie J.N.
      • Hickey E.
      • Weber L.A.
      • Anderson C.W.
      ,
      • Stokoe D.
      • Engel K.
      • Campbell D.G.
      • Cohen P.
      • Gaestel M.
      ), αB-crystallin by MAPKAPK-2, p44/p42 MAPK and PKA (
      • Kato K.
      • Ito H.
      • Kamei K.
      • Inaguma Y.
      • Iwamoto I.
      • Saga S.
      ,
      • Voorter C.E.
      • de Haard-Hoekman W.A.
      • Roersma E.S.
      • Meyer H.E.
      • Bloemendal H.
      • de Jong W.W.
      ), and HSP20 by PKG/PKA (
      • Beall A.
      • Bagwell D.
      • Woodrum D.
      • Stoming T.A.
      • Kato K.
      • Suzuki A.
      • Rasmussen H.
      • Brophy C.M.
      ). It may be speculated that the involvement of differential signaling pathways in phosphorylation of sHSPs is related to the control of the biological activity of these proteins or of the whole complexes.
      Despite considerable efforts, the functions of sHSPs are only poorly understood. As far as has been tested, sHSPs have the ability to protect cells from adverse conditions by preventing both apoptosis and necrosis (
      • Arrigo A.-P.
      • Landry J.
      ). Although not understood in detail, it is likely that their stress-protecting properties are related to their in vitro chaperone-like activity (
      • Rogalla T.
      • Ehrnsperger M.
      • Preville X.
      • Kotlyarov Z.
      • Lutsch G.
      • Ducasse C.
      • Paul C.
      • Wieske M.
      • Arrigo A.-P.
      • Buchner J.
      • Gaestel M.
      ) which may include their ability to stabilize and rearrange cytoskeletal elements (
      • Schäfer C.
      • Clapp P.
      • Welsh M.J.
      • Benndorf R.
      • Williams J.A.
      ,
      • Lavoie J.N.
      • Lambert H.
      • Hickey E.
      • Weber L.A.
      • Landry J.
      ,
      • Piotrowicz R.S.
      • Levin E.G.
      ,
      • Lavoie J.N.
      • Hickey E.
      • Weber L.A.
      • Landry J.
      ). There are now many reports available describing the association of αB-crystallin, HSP27, and HSP20 with cytoskeletal elements such as actin, desmin, vimentin, laminin, and others (
      • Benndorf R.
      • Hayess K.
      • Ryazantsev S.
      • Wieske M.
      • Behlke J.
      • Lutsch G.
      ,
      • Wang K.
      • Spector A.
      ,
      • Nicoll I.D.
      • Quinlan R.A.
      ,
      • Perng M.D.
      • Muchowski P.J.
      • van den IJssel P.
      • Wu G.J.S.
      • Hutcheson A.M.
      • Clark J.I.
      • Quinlan R.A.
      ,
      • Lavoie J.N.
      • Hickey E.
      • Weber L.A.
      • Landry J.
      ). Frequently, this association was found to stabilize the cytoskeletal structures, e.g. αB-crystallin and HSP27 stabilize actin filaments (
      • Wang K.
      • Spector A.
      ,
      • Lavoie J.N.
      • Lambert H.
      • Hickey E.
      • Weber L.A.
      • Landry J.
      ,
      • Lavoie J.N.
      • Hickey E.
      • Weber L.A.
      • Landry J.
      ). It is not clear how the actin-stabilizing property of HSP27 may be related to its actin barbed end capping activity (which inhibits polymerization of actin in vitro) (
      • Benndorf R.
      • Hayess K.
      • Ryazantsev S.
      • Wieske M.
      • Behlke J.
      • Lutsch G.
      ,
      • Miron T.
      • Vancompernolle K.
      • Vandekerckhove J.
      • Wilchek M.
      • Geiger B.
      ). However, the fact that stabilization of the microfilamentsin vivo obviously requires phosphorylated HSP27 (or the corresponding D forms) appears to be consistent with the fact thatin vitro phosphorylated HSP27 or derived peptides have reduced or absent actin polymerization inhibiting activity (
      • Schäfer C.
      • Clapp P.
      • Welsh M.J.
      • Benndorf R.
      • Williams J.A.
      ,
      • Benndorf R.
      • Hayess K.
      • Ryazantsev S.
      • Wieske M.
      • Behlke J.
      • Lutsch G.
      ,
      • Lavoie J.N.
      • Lambert H.
      • Hickey E.
      • Weber L.A.
      • Landry J.
      ,
      • Schneider G.B.
      • Hamano H.
      • Cooper L.F.
      ,
      • Wieske M.
      • Benndorf R.
      • Behlke J.
      • Dölling R.
      • Grelle G.
      • Bielka H.
      • Lutsch G.
      ). Concerning the possible function of HSP22, no definitive conclusions can be drawn with the available data, although its function might be to bind to and modulate the activity of HSP27.

      Acknowledgments

      We thank Lee A. Weber for hHSP27 cDNA Bluescript plasmids, Chenghua Liu for initial help with the yeast two-hybrid system, Jeff Ballew for useful advice and discussions about cloning techniques, and Richard Ransom and Conrad Benndorf for excellent technical assistance. We thank Kate Noon for helpful discussions on interpretation of mass spectra. We also thank the Sequencing Core of the University of Michigan Medical School for skillful work.

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