alpha-thrombin rapidly induces tyrosine phosphorylation of a novel, 74-78-kDa stress response protein(s) in lung fibroblast cells.
We demonstrated previously that exposure of CCL39 lung fibroblasts to alpha-thrombin rapidly inhibits interleukin 6-induced tyrosine phosphorylation of signal transducers and activators of transcription 3 (Stat3). While studying the cross-talk between alpha-thrombin and interleukin 6, we observed that the phospho-specific (tyrosine) anti-Stat3 antibody specifically cross-reacted with a 74-78-kDa protein(s) in alpha-thrombin-treated cells. In this study, we demonstrate that in alpha-thrombin-treated CCL39 cells, the 74-78-kDa protein(s) rapidly undergoes tyrosine phosphorylation. The phosphorylation by alpha-thrombin was detected as early as 5 min and reached a maximum at 15 min; however, low levels were present at 2 h. alpha-Thrombin receptor agonist peptide (SFLLRN) induced its tyrosine phosphorylation, suggesting that alpha-thrombin mediates the effects via protease-activated receptor type 1. Anti-Stat3 antibodies specific to different regions of Stat3 failed to recognize the 74-78-kDa protein(s), suggesting that it is unrelated to Stat3. Cell fractionation experiments showed that it is localized to the cytoplasm. Mass spectrometric analysis of the immunoprecipitated protein showed that the 74-78-kDa protein(s) is related to glucose-regulated protein 75 (GRP-75), a member of the heat shock/stress-response protein family. Consistent with these data, we observed tyrosine phosphorylation of GRP-75 in alpha-thrombin-treated cells. Exposure of cells to pervanadate, a stress-inducing agent, stimulated its tyrosine phosphorylation; however, cytokines and growth factors were ineffective. This is the first report of tyrosine phosphorylation of GRP-75-related stress protein(s) by alpha-thrombin and suggests that this pathway may contribute to the ability of alpha-thrombin to prevent apoptosis in cells exposed to stress or in the injured tissue.
Bhat
G Jayarama
GJ
Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA. Jayarama.gunaje@ttuhsc.edu
Samikkannu
Thangavel
T
Thomas
Jason J
JJ
Thekkumkara
Thomas J
TJ
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D016207:Cytokines / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D047468:Immunoprecipitation; D015850:Interleukin-6 / Q000378:metabolism; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D050796:STAT3 Transcription Factor; D013347:Subcellular Fractions / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D016207:Cytokines; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D015850:Interleukin-6; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D050796:STAT3 Transcription Factor; D015534:Trans-Activators; C018551:glucose-regulated proteins; C076715:pervanadate; C082835:thrombin receptor peptide (42-47); D014638:Vanadates; D014443:Tyrosine; D013917:Thrombin