title: p38 MAP kinase and MAPKAP kinases MK2/3 cooperatively phosphorylate epithelial keratins.
abstract: The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-Ser(73) is catalyzed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38 inhibitors on K8-Ser(73) phosphorylation also demonstrated reduced phosphorylations of keratins K18-Ser(52) and K20-Ser(13) but not of K8-Ser(431) or K18-Ser(33). Interestingly, K18-Ser(52) and K20-Ser(13) are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-Ser(13) and K18-Ser(52) are inhibited by small molecule inhibitors of both p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-Ser(13) phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-Ser(13) phosphorylation between the ileum of wild-type and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.
authors: From the Institute of Biochemistry, Hannover 30625, Germany.
authors: Manoj B
authors: MB
authors: Menon
authors: From the Institute of Biochemistry, Hannover 30625, Germany.
authors: Jessica
authors: J
authors: Schwermann
authors: Department of Gastroenterology, Hepatology, and Endocrinology, Hannover 30625, Germany.
authors: Anurag Kumar
authors: AK
authors: Singh
authors: Proteome Center Tübingen, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen 72076, Germany.
authors: Mirita
authors: M
authors: Franz-Wachtel
authors: Institute of Immunology, Medical School Hannover, Hannover 30625, Germany.
authors: Oliver
authors: O
authors: Pabst
authors: Department of Gastroenterology, Hepatology, and Endocrinology, Hannover 30625, Germany.
authors: Ursula
authors: U
authors: Seidler
authors: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109.
authors: M Bishr
authors: MB
authors: Omary
authors: From the Institute of Biochemistry, Hannover 30625, Germany.
authors: Alexey
authors: A
authors: Kotlyarov
authors: From the Institute of Biochemistry, Hannover 30625, Germany. Electronic address: gaestel.matthias@mh-hannover.de.
authors: Matthias
authors: M
authors: Gaestel
mesh_terms: D000818:Animals; D002460:Cell Line; D007082:Ileum / Q000201:enzymology*; D007413:Intestinal Mucosa / Q000201:enzymology*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007633:Keratins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D007633:Keratins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C098416:MAP-kinase-activated kinase 3