Keratin 8 phosphorylation by p38 kinase regulates cellular keratin filament reorganization: modulation by a keratin 1-like disease causing mutation.
Keratin 8 (K8) serine 73 occurs within a relatively conserved type II keratin motif ((68)NQSLLSPL) and becomes phosphorylated in cultured cells and organs during mitosis, cell stress, and apoptosis. Here we show that Ser-73 is exclusively phosphorylated in vitro by p38 mitogen-activated protein kinase. In cells, Ser-73 phosphorylation occurs in association with p38 kinase activation and is inhibited by SB203580 but not by PD98059. Transfection of K8 Ser-73 --> Ala or K8 Ser-73 --> Asp with K18 generates normal-appearing filaments. In contrast, exposure to okadaic acid results in keratin filament destabilization in cells expressing wild-type or Ser-73 --> Asp K8, whereas Ser-73 --> Ala K8-expressing cells maintain relatively stable filaments. p38 kinase associates with K8/18 immunoprecipitates and binds selectively with K8 using an in vitro overlay assay. Given that K1 Leu-160 --> Pro ((157)NQSLLQPL --> (157)NQSPLQPL) leads to epidermolytic hyperkeratosis, we tested and showed that the analogous K8 Leu-71 --> Pro leads to K8 hyperphosphorylation by p38 kinase in vitro and in transfected cells, likely due to Ser-70 neo-phosphorylation, in association with significant keratin filament collapse upon cell exposure to okadaic acid. Hence, K8 Ser-73 is a physiologic phosphorylation site for p38 kinase, and its phosphorylation plays an important role in keratin filament reorganization. The Ser-73 --> Ala-associated filament reorganization defect is rescued by a Ser-73 --> Asp mutation. Also, disease-causing keratin mutations can modulate keratin phosphorylation and organization, which may affect disease pathogenesis.
Ku
Nam-On
NO
Department of Medicine, and Geriatric Research, Education and Clinical Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, USA.
Azhar
Salman
S
Omary
M Bishr
MB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D006801:Humans; D053533:Keratin-8; D007633:Keratins / Q000737:chemistry / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases
C426203:KRT8 protein, human; D053533:Keratin-8; D007633:Keratins; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases