ISSN:
0886-1544
Keywords:
calyculin A
;
phosphatase 1/2A
;
intermediate filament disruption
;
actomyosin contraction
;
C-kinase
;
intermediate filament protein kinase
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
Cytokeratin and vimentin intermediate filaments (IFs) possess relatively stable polymeric properties which can be affected by phosphorylation. The present study, using cultures of thyroid epithelial cells, shows by indirect immunofluorescence that these cells contain both keratin tonofilament and vimentin IF complexes. Immunoblots of Triton X-100 insoluble cytoskeletal fractions show vimentin, and ∼52 kDa type II and 40/38 kDa type I keratins. Under “basal” conditions, following prelabeling of cells with [32PO4], vimentin is not significantly phosphorylated, while both type II and I keratins are phosphorylated. Treatment of cells for 20 min with 1 mM dbcAMP or 0.4 μM 12-O-tetradecanoylphorbol-13-acetate (TPA), to stimulate protein kinase A and C, respectively, has no effect on either the phosphorylation state or cytoplasmic filament integrity of vimentin. However, while dbcAMP also does not affect keratin filaments, TPA increases both type II and I phosphorylation ∼3-fold, and concomitantly disrupts tonofilament complexes associated with the nucleus, cytoplasm, and desmosomes. TPA-treated cells also show dramatic shape changes and protrusive activity. Tryptic peptide mappings show phosphorylations of at least 6 and ∼2 additional sites for type II and I keratins, respectively, vs. [32P]-peptides from control cells. Treatment of [32PO4]-labeled cells with 0.4 μM calyculin A to inhibit types 1 and 2A phosphatase activity causes hyperphosphorylation of both vimentin and keratin, disruption of IF complexes, and actomyosin/cell contraction within 20 min. Quantitatively, ∼50% of the type II/I keratin hyperphosphorylations are at some sites apparently also phosphorylated after TPA treatment. Thus, in these cells, IFs are specifically and differentially affected and regulated by the activity of several kinases. © 1993 Wiley-Liss, Inc.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/cm.970260407
