doi: 10.1161/01.RES.0000056967.51841.21
© 2003 American Heart Association, Inc.
Editorials |
Serine Phosphorylation and Suppression of Apoptosis by the Small Heat Shock Protein 
B-Crystallin
From the Department of Molecular and Cellular Pharmacology, University of Miami Medical Center, Miami, Fla.
Correspondence to Keith A. Webster, Dept of Molecular and Cellular Pharmacology, University of Miami Medical Center, 1600 NW 10th Ave, RMSB 6038, Miami, FL 33136. E-mail kwebster{at}chroma.med.miami.edu
Key Words: apoptosis • p38 • mitogen-activated protein kinase • crystallin • small heat shock protein
A poptotic death of cardiac myocytes is a central feature of ischemic heart disease and a prime target for therapeutic intervention. Multiple stress factors are associated with ischemic stress and a battery of intrinsic pathways work to attenuate damage. These include antioxidants, antiapoptotic factors such as Bcl-2 proteins, and endogenous caspase inhibitors such as ARC (see review1). 
B-crystallin is a relative newcomer to this defensive arsenal; it has strong antiapoptotic properties and, when fully induced, may constitute as much as 5% of total cardiac myocyte protein. As such, B-crystallin may have critical structural as well as protective functions in the heart. The signaling pathways that regulate B-crystallin activity and the molecular mechanism of protection are not understood and some aspects of these are presently controversial. In this issue of Circulation Research, Morrison et al2 describe the involvement of a critical serine residue at position 59 of the B-crystallin protein that is targeted by the p38ß-MAPKAP-K2 pathway during ischemic stress. They show that phosphorylation of this serine may be the key step for the activation and regulation of the antiapoptotic function(s) of B-crystallin during ischemia.
Crystallin Family Proteins
The crystallins are a family of dual function proteins responsible for the transparent and refractive properties of the eye lens. At least 11 vertebrate crystallins have been described and several of these have secondary chaperoning and antioxidant activities that serve to protect the lens from light-induced oxidative stress (see reviews3,4). B-crystallin is also a small heat shock protein (smHSP) structurally related to other smHSPs including A-crystallin, hsp27, hsp20, hsp22, myotonic dystrophy protein kinase binding protein, and HSPB3.5,6 All of these except A-crystallin are abundantly expressed in muscle where the chaperone activity plays a key survival role in response to stress. B-crystallin is also strongly expressed lung, kidney, brain, and heart. It is induced during myogenic differentiation and is first detected in the heart during development. The selective expression of B-crystallin in muscle is probably mediated by E-box and SRE motifs contained in the B-crystallin promoter that bind the myogenic factors MyoD and MEF-2, respectively.7 B-crystallin monomers (
25 kDa) aggregate into multimeric units of >600 kDa, and these aggregates may be necessary for the chaperoning functions.7 It has been proposed that chaperoning by B-crystallin can stabilize myofilament proteins through selective interactions with desmin, titin, and nebulette.9,10 Ectopic overexpression of B-crystallin protects cardiac myocytes from ischemic damage.4,9,11
Mechanism of Action and Molecular Regulation of B-Crystallin
Multiple stimuli responding to thermal, osmotic, ischemic, and oxidative stress induce and activate B-crystallin through both transcriptional and posttranslational pathways.12 Ischemia promotes the translocation of B-crystallin from diffuse locations in the myoplasm to defined structures in the sarcomere (Z-band) and nucleus, and this translocation may be key to protection.9 Translocation is transient and correlates closely with changes in the phosphorylation and perhaps level of oligomerization of B-crystallin units.8,13 Phosphorylation appears to promote dissociation of B-crystallin oligomers and different groups have associated this with decreased or increased chaperoning activity and cytoprotection.8,9 At least part of the chaperoning functions of B-crystallin is associated with conserved C-terminal peptides that are located on the exterior surface of the oligomeric structures. Mutation of the 2 C-terminal lysine residues to glycine reduces oligomer size by >50% and eliminates protection of cardiac myocytes during ischemia.13
Regulation by Serine-Phosphorylation
The influence of phosphorylation and the structure-function relationships of B-crystallin are controversial and are subjects of the article by Morrison et al.2 In their article, Morrison et al provide new information into how B-crystallin activity may be regulated during osmotic and ischemic stress. They demonstrate that a single serine residue at position 59 that is a target for the p38ß-MAPKAP-2K pathway may be entirely responsible for the activation of B-crystallin during ischemia. This result appears to be at variance with other reports on the effects of serine phosphorylation in other models.
Negative Regulation of smHSPs by Serine Phosphorylation in COS and Glioma Cells
Hsp27 is closely related to B-crystallin, and like B-crystallin, it is phosphorylated by p38-MAPK and protein kinase C pathways in response to stress and growth factors.14 Phosphorylation of hsp27 or pseudophosphorylation by replacement of the target serines with aspartate or glutamate causes dissociation of hsp27 oligomers and reduced stress tolerance.15,16 Under similar stress conditions B-crystallin is phosphorylated on three serine residues including Ser-19, Ser-45, and Ser-59; Ser-45 and Ser-59 are substrates for the p44/42 ERK-MAPK and p38-MAP-KAP-2 pathways, respectively.17 Phosphorylation of these B-crystallin serine residues in COS and glioma cells was reported to mediate reduced oligomerization and decreased chaperone activity and thermotolerance.15,16
Negative Regulation by Serine Phosphorylation in Skeletal Muscle
Native B-crystallin plays an important antiapoptotic role during myogenic differentiation and development.5,6 This is reflected in several settings: a missense mutation of B-crystallin (R120G) causes autosomal dominant myopathy with disruption of myofibrils and accumulation of desmin-B-crystallin aggregates in the degenerating muscle of affected individuals. The R120G mutant is severely compromised in its chaperone activity. B-crystallin protects skeletal myocytes against TNF- and DNA damage–induced apoptosis by inhibiting the processing and activation of caspase 3.5,6 Ectopic expression of wild-type B-crystallin but not the R120G mutant conveyed antiapoptosis to differentiating myocytes.5,6 Importantly, ectopic expression of a pseudophosphorylation B-crystallin mutant with the 3 target serine residues substituted for asparagine was completely devoid of antiapoptotic activity. Neither the R120G nor the pseudophosphorylation B-crystallin mutant was able to inhibit caspase 3. In these studies, it was concluded that serine phosphorylation at positions 19, 45, and 59 inactivated B-crystallin possibly by dissociating aggregates.
Positive Regulation by Serine-Phosphorylation in Cardiac Myocytes
In contrast to reports that the antiapoptotic functions of B-crystallin are inactivated by serine phosphorylation, Morrison et al2 report the opposite effect. In cardiac myocytes subjected to osmotic or ischemic stress, these authors demonstrate that rather than mediating a negative regulation, phosphorylation of Ser-59 is actually required to activate B-crystallin. Caspase 3 again appears to be the target and phosphorylation of Ser-19 and Ser-45 were neutral in these assays. Morrison et al propose that Ser-59 is selectively targeted in cardiac myocytes by the MKK6-p38ß-MAPKAP-K2 kinase cascade that is initiated by osmotic and ischemic stress12 (see Figure). By substituting each of the B-crystallin serine resides individually or in combination with either alanine to block phosphorylation or glutamine to mimic constitutive phosphorylation, Morrison et al defined the contribution of each site to B-crystallin activity. They demonstrate that B-crystallin is inactive when Ser-59 is substituted by Ala, and fully active when Ser-59 is replaced by Glu. Neonatal cardiac myocytes expressing the Ala substitution at Ser-59 were 3-fold more susceptible to osmotic and ischemic stress, whereas myocytes expressing the Glu substitution were 3-fold more resistant. In each case, susceptibility to apoptosis correlated with caspase 3 activity.
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All investigators agree that B-crystallin can provide powerful antiapoptotic protection against multiple stresses. At least 2 pathways appear to account for this: (1) ischemia in cardiac myocytes or growth factor deprivation in differentiating skeletal myocytes activate B-crystallin expression and promote translocation to myofilament and nuclear structures where the chaperone functions confer myofilament stabilization; and (2) the same stress conditions that promote translocation also favor increased association of B-crystallin with other cellular proteins, including apoptosis regulators such as caspase 3 (p24). The translocation and binding activities may be related and both probably contribute to cytoprotection and suppression of apoptosis. Precisely how these activities are differentially regulated by serine phosphorylation is not clear. As shown by Morrison et al,2 Ser-59 phosphorylation is required to protect cardiac myocytes from ischemia, but the same phosphorylation appears to inhibit this function in differentiating skeletal myocytes.5,6 This is perplexing because caspase 3 was reported to be the target in both instances. Clearly the signaling cascades that are activated by ischemic stress are different from those that are active during myogenic differentiation and the outcome is determined by the net effect of multiple cross-reacting pathways. It may also be assumed that Ser-19 and Ser-45 phosphorylation will contribute to function under some circumstances. Two previous studies support a positive role of serine phosphorylation (see references 27 and 36 in Morrison et al2).
The contribution of oligomerization to chaperone activity of B-crystallin is also not resolved. Morrison et al2 suggest that B-crystallin monomers may be the preferred chaperones whereas Ito et al8 and Martin et al13 conclude the opposite. It seems possible that the state of oligomerization of B-crystallin is secondary to other factors that regulate function. Although further studies are required to explain these controversies, Morrison et al have made significant progress towards establishing a full signaling pathway for cytoprotection by B-crystallin in ischemic cardiac myocytes.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
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2. Morrison LE, Hoover HE, Thuerauf DJ, Glembotski CC. Mimicking phosphorylation of B-Crystallin on serine-59 is necessary and sufficient to provide maximal protection of cardiac myocytes from apoptosis. Circ Res. 2003; 92: 203–211.
3. Piatigorsky J. Multifunctional lens crystallins and corneal enzymes. More than meets the eye. Ann N Y Acad Sci. 1998; 842: 7–15.[CrossRef][Medline] [Order article via Infotrieve]
4. Dillmann WH. Small heat shock proteins and protection against injury. Ann N Y Acad Sci. 1999; 874: 66–68.[CrossRef][Medline] [Order article via Infotrieve]
5. Kamradt MC, Chen F, Sam S, Cryns VL. The small heat shock protein B-crystallin negatively regulates apoptosis during myogenic differentiation by inhibiting caspase-3 activation. J Biol Chem. 2002; 277: 38731–38736.
6. Kamradt MC, Chen F, Cryns VL. The small heat shock protein B-crystallin negatively regulates cytochrome c- and caspase-8-dependent activation of caspase-3 by inhibiting its autoproteolytic maturation. J Biol Chem. 2001; 276: 16059–16063.
7. Sugiyama Y, Suzuki A, Kishikawa M, Akutsu R, Hirose T, Waye MM, Tsui SK, Yoshida S, Ohno S. Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation. J Biol Chem. 2000; 275: 1095–1104.
8. Ito H, Kamei K, Iwamoto I, Inaguma Y, Nohara D, Kato K. Phosphorylation-induced change of the oligomerization state of B-crystallin. J Biol Chem. 2001; 276: 5346–5352.
9. Golenhofen N, Ness W, Koob R, Htun P, Schaper W, Drenckhahn D. Ischemia-induced phosphorylation and translocation of stress protein B-crystallin to Z lines of myocardium. Am J Physiol. 1998; 274: H1457–H1464.[Medline]
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11. Ray PS, Martin JL, Swanson EA, Otani H, Dillmann WH, Das DK. Transgene overexpression of B crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. FASEB J. 2001; 15: 393–402.
12. Hoover HE, Thuerauf DJ, Martindale JJ, Glembotski CC. B-crystallin gene induction and phosphorylation by MKK6-activated p38: a potential role for B-crystallin as a target of the p38 branch of the cardiac stress response. J Biol Chem. 2000; 275: 23825–23833.
13. Martin JL, Bluhm WF, He H, Mestril R, Dillmann WH. Mutation of COOH-terminal lysines in overexpressed B-crystallin abrogates ischemic protection in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2002; 283: H85–H91.
14. Kato K, Hasegawa K, Goto S, Inaguma Y. Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. J Biol Chem. 1994; 269: 11274–11278.
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16. Rogalla T, Ehrnsperger M, Preville X, Kotlyarov A, Lutsch G, Ducasse C, Paul C, Wieske M, Arrigo AP, Buchner J, Gaestel M. Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor by phosphorylation. J Biol Chem. 1999; 274: 18947–18956.
17. Kato K, Ito H, Kamei K, Inaguma Y, Iwamoto I, Saga S. Phosphorylation of B-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation. J Biol Chem. 1998; 273: 28346–28354.
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