Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle.

The 27-kDa heat shock protein (HSP27) is expressed in a variety of tissues in the absence of stress and is thought to regulate actin filament dynamics, possibly by a phosphorylation/dephosphorylation mechanism. HSP27 has also been suggested to be involved in contraction of intestinal smooth muscle. We have investigated phosphorylation of HSP27 in airway smooth muscle in response to the muscarinic agonist carbachol. Carbachol increased32P incorporation into canine tracheal HSP27 and induced a shift in the distribution of charge isoforms on two-dimensional gels to more acidic, phosphorylated forms. The canine HSP27 amino acid sequence includes three serine residues corresponding to sites in human HSP27 known to be phosphorylated by mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2. To determine whether muscarinic receptors are coupled to a "stress response" pathway in smooth muscle culminating in phosphorylation of HSP27, we assayed MAPKAP kinase-2 activity and tyrosine phosphorylation of p38 mitogen-activated protein (MAP) kinase, the enzyme thought to activate MAPKAP kinase-2. Recombinant canine HSP27 expressed in Escherichia coli was a substrate for MAPKAP kinase-2 in vitro as well as a substrate for endogenous smooth muscle HSP27 kinase, which was activated by carbachol. Carbachol also increased tyrosine phosphorylation of p38 MAP kinase. SB-203580, an inhibitor of p38 MAP kinases, reduced activation of endogenous HSP27 kinase activity and blocked the shift in HSP27 charge isoforms to acidic forms. We suggest that HSP27 in airway smooth muscle, in addition to being a stress response protein, is phosphorylated by a receptor-initiated signaling cascade involving muscarinic receptors, tyrosine phosphorylation of p38 MAP kinase, and activation of MAPKAP kinase-2.

[1]  J. Landry,et al.  Transient activation of a distinct serine protein kinase is responsible for 27-kDa heat shock protein phosphorylation in mitogen-stimulated and heat-shocked cells. , 1993, The Journal of biological chemistry.

[2]  M. Kaminski,et al.  Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin. , 1991, Biochemical and biophysical research communications.

[3]  G. Stein,et al.  Sequence and organization of genes encoding the human 27 kDa heat shock protein. , 1986, Nucleic acids research.

[4]  M. Gaestel,et al.  Small heat shock proteins are molecular chaperones. , 1993, The Journal of biological chemistry.

[5]  L. Adam,et al.  Activation of mitogen-activated protein kinase in porcine carotid arteries. , 1995, Circulation research.

[6]  R. Sha’afi,et al.  Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation. , 1996, Blood.

[7]  J. Landry,et al.  Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization. , 1993, The Journal of biological chemistry.

[8]  Howard M. Goodman,et al.  High resolution two-dimensional electrophoresis of basic as well as acidic proteins , 1977, Cell.

[9]  J. Nick,et al.  Interleukin-8 Regulation of the Ras/Raf/Mitogen-activated Protein Kinase Pathway in Human Neutrophils (*) , 1996, The Journal of Biological Chemistry.

[10]  J. Sellers,et al.  Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle. , 1996, The Journal of physiology.

[11]  Michel Morange,et al.  A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins , 1994, Cell.

[12]  M. Gaestel,et al.  Interleukin-1-induced intracellular signaling pathways converge in the activation of mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 and the subsequent phosphorylation of the 27-kilodalton heat shock protein in monocytic cells. , 1994, Molecular pharmacology.

[13]  W. Gerthoffer,et al.  Cloning and sequencing of a cDNA encoding the canine HSP27 protein. , 1995, Gene.

[14]  P. Sugden,et al.  Cellular Stresses Differentially Activate c-Jun N-terminal Protein Kinases and Extracellular Signal-regulated Protein Kinases in Cultured Ventricular Myocytes (*) , 1995, The Journal of Biological Chemistry.

[15]  Philip R. Cohen,et al.  A comparison of the substrate specificity of MAPKAP kinase‐2 and MAPKAP kinase‐3 and their activation by cytokines and cellular stress , 1996, FEBS letters.

[16]  Philip R. Cohen,et al.  SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin‐1 , 1995, FEBS letters.

[17]  H. Kawasaki,et al.  Evidence for Multiple Activators for Stress-activated Protein Kinases/c-Jun Amino-terminal Kinases. , 1995, The Journal of Biological Chemistry.

[18]  N. Qian,et al.  Involvement of Ras and Raf in the Gi-coupled acetylcholine muscarinic m2 receptor activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase. , 1993, The Journal of biological chemistry.

[19]  J. Landry,et al.  Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. , 1993, The Journal of biological chemistry.

[20]  A. Ridley,et al.  The small GTP-binding protein rac regulates growth factor-induced membrane ruffling , 1992, Cell.

[21]  R. Moreland,et al.  Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery. , 1995, The American journal of physiology.

[22]  W. Gerthoffer,et al.  Activation of MAP kinases in airway smooth muscle. , 1997, The American journal of physiology.

[23]  E. Levin,et al.  Identification of a protein transiently phosphorylated by activators of endothelial cell function as the heat-shock protein HSP27. A possible role for protein kinase C. , 1992, The Biochemical journal.

[24]  R. Kramer,et al.  Thrombin Induces Activation of p38 MAP Kinase in Human Platelets (*) , 1995, The Journal of Biological Chemistry.

[25]  L. Adam,et al.  Identification of mitogen‐activated protein kinase phosphorylation sequences in mammalian h‐Caldesmon , 1993, FEBS letters.

[26]  David Stokoe,et al.  Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins , 1992, FEBS letters.

[27]  J. Strahler,et al.  Activation of MAP kinase and translocation with HSP27 in bombesin-induced contraction of rectosigmoid smooth muscle. , 1995, The American journal of physiology.

[28]  L Bibbs,et al.  A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. , 1994, Science.

[29]  J. Behlke,et al.  Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity. , 1994, The Journal of biological chemistry.

[30]  C. Anderson,et al.  Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II. , 1992, The Journal of biological chemistry.

[31]  J. Hsuan,et al.  Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of hsp27 , 1994, Cell.

[32]  Anne J. Ridley,et al.  The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors , 1992 .

[33]  J. Landry,et al.  Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27. , 1997, Journal of cell science.