Phosphorylation of the Small Heat Shock-related Protein, HSP20, in Vascular Smooth Muscles Is Associated with Changes in the Macromolecular Associations of HSP20

Brophy, Colleen M.; Dickinson, Mary; Woodrum, David A.

doi:10.1074/jbc.274.10.6324

Cited by 67 publications

(66 citation statements)

References 18 publications

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“…Because acetylation tends to occur in large macromolecular complexes (3,4) it is important to note that Hsp20 and Hsp27 also exist in smooth muscle cells in macromolecular complexes ranging from dimers to multimers of 500 -700 kDa (34). These complexes are dynamically regulated by protein kinases such as PKA, which phosphorylates serine 16 within Hsp20 (26 -28).…”

Section: Discussionmentioning

confidence: 99%

Acetylation of Heat Shock Protein 20 (Hsp20) Regulates Human Myometrial Activity

Karolczak-Bayatti

Sweeney

Cheng

et al. 2011

Journal of Biological Chemistry

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Phosphorylation of heat shock protein 20 (Hsp20) by protein kinase A (PKA) is now recognized as an important regulatory mechanism modulating contractile activity in the human myometrium. Thus agonists that stimulate cyclic AMP production may cause relaxation with resultant beneficial effects on pathologies that affect this tissue such as the onset of premature contractions prior to term. Here we describe for the first time that acetylation of Hsp20 is also a potent post-translational modification that can affect human myometrial activity. We show that histone deacetylase 8 (HDAC8) is a non-nuclear lysine deacetylase (KDAC) that can interact with Hsp20 to affect its acetylation. Importantly, use of a selective linkerless hydroxamic acid HDAC8 inhibitor increases Hsp20 acetylation with no elevation of nuclear-resident histone acetylation nor marked global gene expression changes. These effects are associated with significant inhibition of spontaneous and oxytocin-augmented contractions of ex vivo human myometrial tissue strips. A potential molecular mechanism by which Hsp20 acetylation can affect myometrial activity by liberating cofilin is described and further high-lights the use of specific effectors of KDACs as therapeutic agents in regulating contractility in this smooth muscle.In developed countries premature birth prior to 37 weeks gestation accounts for nearly 75% of newborn deaths and is related to a high risk for survivors of long-term physical or mental disability (1). There are still no effective and safe therapeutic treatments for decreasing the incidence of premature deliveries. Accordingly, an increased understanding of the molecular mechanisms underlying myometrial activity is required to aid in the development of new strategies for the treatment of premature labor. To this end, recent evidence indicates a novel role for post-translational modification by acetylation in regulating myometrial activity. This was first highlighted by the class I/II histone deacetylase inhibitors (HDACIs) 3 TSA, VPA, and SBHA effecting relaxations of ϳ60% to spontaneously or oxytocin-mediated contracting human myometrial tissues ex vivo (2). Potential epigenetic events resulting from increased histone acetylation seem to be excluded due to the comparatively short time period observed for contractile inhibition of 20 -60 min. This pointed to the above HDACIs having non-epigenetic effects involving increased acetylation of lysine residues of protein components of the myometrial contractile machinery. This possibility is supported by recent proteomic findings of Kim et al. (3) and Choudhary et al. (4). They, respectively, showed that administration of the HDACIs TSA/Sirtinol to HeLa or SAHA/MS-275 to leukemia MV4 -11 cell cultures resulted in acetylation of a range of non-nuclear proteins. Importantly, proteins involved in regulating the cytoskeletal/filamentous architecture of cells were observed to be acetylated. These included actin, cofilin, 14-3-3 as well as the heat shock family protein member Hsp27.

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Section: Discussionmentioning

confidence: 99%

Acetylation of Heat Shock Protein 20 (Hsp20) Regulates Human Myometrial Activity

Karolczak-Bayatti

Sweeney

Cheng

et al. 2011

Journal of Biological Chemistry

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“…13 In brief, the tissues were homogenized in HEPES buffer (0.5 g tissue/1 mL buffer) at 4°C. The homogenate was centrifuged at 100 000g for 30 minutes at 4°C.…”

Section: Gel Filtrationmentioning

confidence: 99%

“…12,13 Because the small HSPs were predominantly cytosolic proteins, gel filtration was performed on the cytosolic fractions of heart muscle homogenates with superose 6 columns. 13 Fractions from the column were dot-blotted with antibodies against HSP20, ␣B-crystallin, and MKBP. HSP20 and ␣B-crystallin were found in similar fractions that eluted from the column after the catalase standard (MW 232, Figure 3A).…”

Section: Subcellular Localization and Macromolecular Associations Of mentioning

confidence: 99%

“…11 These proteins are often associated in large macromolecular aggregates. 12,13 Heat shock proteins have been implicated in modulating the cellular response to many stressors (heat, chemical, osmotic, oxidative, and mechanical) and in modulating protein-protein interactions. An additional recently described function of heat shock proteins is to modulate the activity of kinases.…”

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confidence: 99%

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Localization, Macromolecular Associations, and Function of the Small Heat Shock–Related Protein HSP20 in Rat Heart

et al. 2003

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Background-The small heat shock proteins HSP20, HSP25, ␣B-crystallin, and myotonic dystrophy kinase binding protein (MKBP) may regulate dynamic changes in the cytoskeleton. For example, the phosphorylation of HSP20 has been associated with relaxation of vascular smooth muscle. This study examined the function of HSP20 in heart muscle. Methods and Results-Western blotting identified immunoreactive HSP20, ␣B-crystallin, and MKBP in rat heart homogenates. Subcellular fractionation demonstrated that HSP20, ␣B-crystallin, and MKBP were predominantly in cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and ␣B-crystallin were associated in an aggregate of Ϸ200 kDa, and ␣B-crystallin coimmunoprecipitated with HSP20. Immunofluorescence microscopy demonstrated that the pattern of HSP20, ␣B-crystallin, and actin staining was predominantly in transverse bands. Treatment with sodium nitroprusside led to increases in the phosphorylation of HSP20, as determined with 2-dimensional immunoblots. Incubation of transiently permeabilized myocytes with phosphopeptide analogues of HSP20 led to an increase in the rate of shortening. The increased shortening rate was associated with an increase in the rate of lengthening and a more rapid decay of the calcium transient. Conclusions-HSP20 is associated with ␣B-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20 increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening.

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“…Expression and ser16 phosphorylation of HSP20 have been strongly associated with relaxation in smooth muscle (3,5,9) and treatment of CSMC with the relaxant neuropeptide, VIP, caused significant and substantial phosphorylation of HSP20 by 30 s posttreatment (21). These observations led us to perform studies to determine the effect of expression of recombinant HSP20 mutant on early contraction in CSMC.…”

Section: Characterization Of Rabbit Csmc Transfected With Hsp20 Phospmentioning

confidence: 99%

Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle

Somara

Gilmont

Varadarajan

et al. 2010

American Journal of Physiology-Gastrointestinal and Liver Physi

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Somara S, Gilmont RR, Varadarajan S, Bitar KN. Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle. Am J Physiol Gastrointest Liver Physiol 299: G1164 -G1176, 2010. First published September 9, 2010 doi:10.1152/ajpgi.00479.2009.-Small heat shock proteins HSP27 and HSP20 have been implicated in regulation of contraction and relaxation in smooth muscle. Activation of PKC-␣ promotes contraction by phosphorylation of HSP27 whereas activation of PKA promotes relaxation by phosphorylation of HSP20 in colonic smooth muscle cells (CSMC). We propose that the balance between the phosphorylation states of HSP27 and HSP20 represents a molecular signaling switch for contraction and relaxation. This molecular signaling switch acts downstream on a molecular mechanical switch [tropomyosin (TM)] regulating thin-filament dynamics. We have examined the role of phosphorylation state(s) of HSP20 on HSP27-mediated thin-filament regulation in CSMC. CSMC were transfected with different HSP20 phosphomutants. These transfections had no effect on the integrity of actin cytoskeleton. Cells transfected with 16D-HSP20 (phosphomimic) exhibited inhibition of acetylcholine (ACh)-induced contraction whereas cells transfected with 16A-HSP20 (nonphosphorylatable) had no effect on ACh-induced contraction. CSMC transfected with 16D-HSP20 cDNA showed significant decreases in 1) phosphorylation of HSP27 (ser78); 2) phosphorylation of PKC-␣ (ser657); 3) phosphorylation of TM and CaD (ser789); 4) ACh-induced phosphorylation of myosin light chain; 5) ACh-induced association of TM with HSP27; and 6) ACh-induced dissociation of TM from caldesmon (CaD). We thus propose the crucial physiological relevance of molecular signaling switch (phosphorylation state of HSP27 and HSP20), which dictates 1) the phosphorylation states of TM and CaD and 2) their dissociations from each other.relaxation; heat shock protein; molecular signaling switch; molecular mechanical switch; smooth muscle contraction AGONIST-INDUCED INCREASES in intracellular Ca 2ϩ concentrations have been implicated in the initiation of smooth muscle contraction (42,46, 54,61). Although phosphorylation of regulatory myosin light chains (MLC 20 ) by activation of Ca 2ϩ / calmodulin-dependent myosin light chain kinase (MLCK) is the key mechanism for initiation of smooth muscle contraction, inactivation of myosin light chain phosphatase (MLCP) is the key mechanism for maintenance of smooth muscle contraction (25). However, maintenance of force upon decrease in intracellular calcium concomitant with decrease in phosphorylation of MLC 20 is associated with thin-filament regulation (18,36,38,64). Thin-filament regulation explains the maintenance of tone at low MLC 20 phosphorylation. Thin-filament regulation modulates the interaction of actin molecules of the thin filament with myosin heads of the thick filament for contraction to occur (10,20,53,65).Relaxation of smooth muscle occurs either as a result of removal of the con...

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Phosphorylation of the Small Heat Shock-related Protein, HSP20, in Vascular Smooth Muscles Is Associated with Changes in the Macromolecular Associations of HSP20

Cited by 67 publications

References 18 publications

Acetylation of Heat Shock Protein 20 (Hsp20) Regulates Human Myometrial Activity

Acetylation of Heat Shock Protein 20 (Hsp20) Regulates Human Myometrial Activity

Localization, Macromolecular Associations, and Function of the Small Heat Shock–Related Protein HSP20 in Rat Heart

Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle

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