14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor.
14-3-3 is a ubiquitous protein family that interacts with several signal transduction kinases. We show that 14-3-3 proteins associate with keratin intermediate filament polypeptides 8 and 18 (K8/18) that are expressed in simple-type epithelia. The association is stoichiometrically significant (> or = one 14-3-3 molecule/keratin tetramer), occurs preferentially with K18, and is phosphorylation- and cell cycle-dependent in that it occurs during S/G2/M phases of the cell cycle when keratins become hyperphosphorylated. Binding of phospho-K8/18 to 14-3-3 can be reconstituted in vitro using recombinant 14-3-3 or using total cellular cytosol. Phosphatase treatment results in dissociation of 14-3-3, and dephosphorylation of phospho-K8/18 prevents reconstitution of the binding. Three cellular keratin subpopulations were analyzed that showed parallel gradients of keratin phosphorylation and 14-3-3 binding. Incubation of 14-3-3 with keratins during or after in vitro filament assembly results in sequestering of additional soluble keratin, only in cases when the keratins were hyperphosphorylated. Our results demonstrate a stoichiometrically significant cell cycle- and phosphorylation-regulated binding of 14-3-3 proteins to K18 and in vitro evidence of a simple epithelial keratin sequestering role for 14-3-3 proteins.
Liao
J
J
VA Palo Alto Health Care System, CA 94304, USA.
Omary
M B
MB
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism; D007633:Keratins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D007633:Keratins; D014446:Tyrosine 3-Monooxygenase