A significant soluble keratin fraction in 'simple' epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility.
We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.
Chou
C F
CF
Palo Alto Veterans Administration Medical Center, CA.
Riopel
C L
CL
Rott
L S
LS
Omary
M B
MB
D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D002460:Cell Line; D003110:Colonic Neoplasms / Q000473:pathology; D004848:Epithelium / Q000737:chemistry*; D006031:Glycosylation; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry; D007399:Interphase; D007633:Keratins / Q000737:chemistry / Q000302:isolation & purification*; D007668:Kidney; D007614:Macropodidae; D008677:Metaphase; D009363:Neoplasm Proteins / Q000032:analysis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012995:Solubility; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D007633:Keratins