Phosphorylation of HSP20 on Serine 157

The following is the abstract of the article discussed in the subsequent letter:

	ABSTRACT

Rembold, Christopher M., Matthew O'Connor, Michael Clarkson, Robert L. Wardle, and Richard A. Murphy. Selected Contribution: HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone. J Appl Physiol 92: 1460-1466, 2001.Cyclic nucleotide-induced relaxation of maximally activated arterial smooth muscle has two phases. 1) The initial relaxation transient is typically characterized by a rapid reduction in force associated with brief reductions in myoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and myosin regulatory light chain (MRLC) phosphorylation on serine (Ser)-19 (Ser¹⁹). 2) The sustained inhibitory response is typically associated with Ser¹⁶ phosphorylation of heat shock protein 20 (HSP20) without sustained reductions in [Ca²⁺]_i or MRLC phosphorylation. We investigated whether the extent of Ser¹⁶-HSP20 phosphorylation quantitatively correlated with the sustained inhibitory response. With addition of nitroglycerin to histamine-stimulated swine carotid media, the initial relaxation transient was associated with a decrease in MRLC phosphorylation without an increase in Ser¹⁶-HSP20 phosphorylation. During the sustained phase of nitroglycerin-induced relaxation and during force redevelopment induced by washout of nitroglycerin in the continued presence of histamine, the level of Ser¹⁶-HSP20 phosphorylation, but not MRLC phosphorylation, correlated with inhibition of force. Forskolin, which increases cAMP concentration, also induced a sustained inhibitory response that was associated with increases in Ser¹⁶-HSP20 phosphorylation without reductions in MRLC phosphorylation levels. Forskolin increased Ser¹⁶-HSP20 phosphorylation to a greater extent and inhibited force more completely than that observed with nitroglycerin. Increases in Ser¹⁶-HSP20 phosphorylation correlated with the degree of force inhibition regardless of whether the relaxation was induced by nitroglycerin or forskolin. These data are consistent with the hypothesis that Ser¹⁶-HSP20 phosphorylation may be a cyclic nucleotide-dependent, yet MRLC phosphorylation-independent, inhibitor of smooth muscle contractile force.

	LETTER

Phosphorylation of HSP20 on Serine 157

To the Editor: It is gratifying to note that the physiological relevance of HSP20 in vascular smooth muscle is receiving increasing attention. We were pleased to note that Rembold et al. (2) successfully repeated experiments that we previously reported (4), suggesting that cyclic nucleotide-dependent phosphorylation of HSP20 on serine 16 inhibits smooth muscle force, independent of myosin light chain phosphorylation. We also demonstrated that cyclic nucleotide-dependent inhibition of force was independent of oxygen consumption. We were somewhat surprised that Dr. Rembold and colleagues did not compare their results with ours. Regarding the phosphorylation of HSP20 on serine 157, this has been well characterized and reported by other investigators (3), and their work should be recognized. However, the serine 157 phosphorylation site is not in the human HSP20 sequence (GenBank assession no. O14558), and we have described only a single phosphorylated isoform of HSP20 in human tissue (1, 4). Thus the physiological relevance of the serine 157 site is questionable.

	REFERENCES

1.   Bergh, CM, Brophy CM, Dransfield DT, Lincoln T, Goldenring JR, and Rasmussen H. Impaired cyclic nucleotide-dependent vasorelaxation in human umbilical artery smooth muscle. Am J Physiol Heart Circ Physiol Heart Circ Physiol 268: H202-H212, 1995[Abstract/Free Full Text].

2.   Rembold, CM, O'Connor M, Clarkson M, Wardle RL, and Murphy RA. Selected Contribution: HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone. J Appl Physiol 91: 1460-1466, 2001[Abstract/Free Full Text].

3.   Wang, Y, Xu A, Pearson RB, and Cooper GJ. Insulin and insulin antagonist evoke phosphorylation of P20 and serine 157 and serine 16, respectively, in rat skeletal muscle. FEBS Lett 462: 25-30, 1999[ISI][Medline].

4.   Woodrum, DA, Brophy CM, Wingard CJ, Beall A, and Rasmussen H. Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. Am J Physiol Heart Circ Physiol Heart Circ Physiol 277: H931-H939, 1999[Abstract/Free Full Text].

Colleen Brophy, Phoenix Veterans Affairs Medical Center Phoenix, Arizona 85012 E-mail: brophy.colleen{at}asu.edu

David Woodrum, Mayo Clinic Rochester, Minnesota 55904 E-mail: dawoodrum{at}aol.com

	REPLY

To the Editor: We sincerely regret the inadvertent omission of a reference to Woodrum et al. (4). However, we did cite an earlier study from Dr. Brophy's laboratory (1) that suggested that phosphorylation of serine 16 in heat shock protein 20 (HSP20) is associated with cyclic nucleotide-mediated relaxation in smooth muscle. Although we used different methodologies to estimate both HSP20 and myosin regulatory light chain phosphorylation, we confirmed their findings. Our paper (3) provides important new information that demonstrates that HSP20 phosphorylation is too slow to account for the rapid decline in tone induced by nitric oxide donors. Falling force, per se, is correlated with a rapid transient myosin regulatory light chain dephosphorylation attributable to both a decrease in Ca²⁺-dependent myosin kinase activity and a transient increase in myosin phosphatase activity (2). HSP20 phosphorylation is proportional to the steady-state maintenance of the reduced force despite a recovery of cross-bridge phosphorylation to pre-nitric oxide donor values.

	FOOTNOTES

10.1152/japplphysiol.00879.2001

	REFERENCES

1.   Beall, A, Bagwell D, Woodrum D, Stoming TA, Kato K, Suzuki A, Rasmussen H, and Brophy CM. The small heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation. J Biol Chem 274: 11344-11351, 1999[Abstract/Free Full Text].

2.   Etter, EF, Eto M, Wardle RL, Brautigan DL, and Murphy RA. Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relation. J Biol Chem 276: 34681-34685, 2001[Abstract/Free Full Text].

3.   Rembold, CM, O'Connor M, Clarkson M, Wardle RL, and Murphy RA. Selected Contribution: HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone. J Appl Physiol 91: 1460-1466, 2001.

4.   Woodrum, DA, Brophy CM, Wingard CJ, Beall A, and Rasmussen H. Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. Am J Physiol Heart Circ Physiol Heart Circ Physiol 277: H931-H939, 1999.

Christopher M. Rembold, Robert L. Wardle, and Richard A. Murphy1 Departments of Internal Medicine (Cardiology) and Molecular Physiology and Biological Physics University of Virginia Health System Charlottesville, Virginia 22908 E-mail: crembold{at}virginia.edu