p38 MAP kinase and MAPKAP kinases MK2/3 cooperatively phosphorylate epithelial keratins.
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.
Menon
Manoj B
MB
From the Institute of Biochemistry, Hannover 30625, Germany.
Schwermann
Jessica
J
From the Institute of Biochemistry, Hannover 30625, Germany.
Singh
Anurag Kumar
AK
Department of Gastroenterology, Hepatology, and Endocrinology, Hannover 30625, Germany.
Franz-Wachtel
Mirita
M
Proteome Center Tübingen, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen 72076, Germany.
Pabst
Oliver
O
Institute of Immunology, Medical School Hannover, Hannover 30625, Germany.
Seidler
Ursula
U
Department of Gastroenterology, Hepatology, and Endocrinology, Hannover 30625, Germany.
Omary
M Bishr
MB
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109.
Kotlyarov
Alexey
A
From the Institute of Biochemistry, Hannover 30625, Germany.
Gaestel
Matthias
M
From the Institute of Biochemistry, Hannover 30625, Germany. Electronic address: gaestel.matthias@mh-hannover.de.
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
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