The molecular basis for the autoregulation of calponin by isoform-specific C-terminal tail sequences.
The three genetic isoforms of calponin (CaP), h1, h2 and acidic, are distinguished mostly by their individual C-terminal tail sequences. Deletion of these sequences beyond the last homologous residue Cys273 increases actin filament association for all three isoforms, indicating a negative regulatory role for the unique tail regions. We have tested this hypothesis by constructing a series of deletion and substitution mutants for all three CaP isoforms. Here we demonstrate that the C-terminal sequences regulate actin association by altering the function of the second actin-binding site, ABS2, in CaP comprised of the three 29-residue calponin repeats. Removal of the inhibitory tail resulted in an increased binding and bundling activity, and caused a prominent re-localization of h2 CaP from the peripheral actin network to the central actin stress fibers in transfected A7r5 smooth muscle cells. Domain-swap experiments demonstrated that the tail sequence of h2 CaP can downregulate cytoskeletal association efficiently in all three CaP isoforms, whereas the tail of the smooth-muscle-specific h1 CaP variant had little effect. Site-directed mutagenesis further revealed that the negatively charged residues within the tail region are essential for this regulatory function. Finally we demonstrate that the tail sequences regulate the second actin-binding site (ABS2) and not the strong actin-binding ABS1 region in CaP.
Burgstaller
Gerald
G
Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
Kranewitter
Wolfgang J
WJ
Gimona
Mario
M
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D007526:Isoelectric Point; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009130:Muscle, Smooth; D009154:Mutation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D020033:Protein Isoforms; D000096985:Calponins