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The molecular basis for the autoregulation of calponin by isoform-specific C-terminal tail sequences

Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria

View larger version (26K): [in a new window]   Fig. 1. (A) The delineated functional and structural domains in calponins. Primary domains are the CH domain, the strong actin-binding site (ABS1), the adjacent triple CLIK repeats harboring the ABS2, and the C-terminal tail. (B) Amino acid exchanges in h2 CaP within the ABS1 region at positions 151 and 156. Note the characteristic h2-type exchanges in the ABS1 region of XCaPH3 from Xenopus laevis. (C) CaP mutants used in this study. All constructs carry the GFP fusion at their respective N-terminal ends. Color coding refers to h1 CaP (pink), h2 CaP (orange), and acidic CaP (brown). The non-functional ABS1 in h2 CaP is shown in gray. Mutant domains are shown as hatched blue boxes.

View larger version (81K): [in a new window]   Fig. 2. Subcellular localization of h1 CaP constructs in A7r5 cells. Both h1 CaP and h1 CaPt localize preferentially to the central portion of the stress fibers. Note the lack of overlap for EGFP (green) and phalloidin (red) signals at the ends of stress fibers, corresponding to sites of focal adhesions (arrows). Disruption of the ABS1 by site-directed mutagenesis (h1ABS1-k.o.) causes a significant change in the localization pattern. This effect is reversed by the additional deletion of the tail (h1t ABS1-k.o.). Top row, actin visualized by Alexa 568 phalloidin; middle row, GFP fluorescence; bottom row, merged images.

View larger version (38K): [in a new window]   Fig. 3. (A) Extraction profiles of h1 CaP. Percentage of non-extractable, ectopically expressed h1 CaP constructs in cultured A7r5 smooth muscle cells at three different ionic conditions. The mutant lacking the entire C-terminal tail sequence (h1t) is retained in the cytoskeletal fraction even at higher KCl concentrations compared with the wild-type h1 CaP. Exchanging the tail in h1 CaP for that of either h2 CaP (h1-h2t) or ac. CaP (h1-act) reduces in both cases the amount of CaP present in the cytoskeletal fraction at all three ionic conditions tested. Introduction of the K151N and R156T mutations into the ABS1 of h1 CaP (h1 ABS1-k.o.) also results in a significant reduction of cytoskeletal association. Additional removal of the tail (h1t ABS1-k.o.) restores the levels of this double h1 CaP mutant in the pellet to levels comparable with that of wild-type h1 CaP. (B) Extraction profiles of h2 CaP. Percentage of non-extractable, ectopically expressed h2 CaP constructs in cultured A7r5 smooth muscle cells at three different ionic conditions. Similar to h1 CaP, a mutant lacking the entire C-terminal tail sequence (h2t) increases the amount of CaP present in the cytoskeletal fraction even at higher KCl concentrations compared with the otherwise easily extractable wild-type h2 CaP. Exchanging the tail in h2 CaP for that of either h1 CaP (h2-h1t) or ac. CaP (h2-act) also increases the amount h2 CaP retained in the cytoskeletal fraction, albeit to lower levels. Note that the strongly acidic tail from ac. CaP is less effective than the neutral h1 tail. Mutations of the C-terminal negatively charged residues h2 ch-k.o. neutralize the overall negative character of the tail and result in an extraction profile similar to that for the h2-h1t and h2 t mutants.

View larger version (97K): [in a new window]   Fig. 4. Subcellular localization of h2 CaP constructs in A7r5 cells. Deletion of the negatively charged tail sequence in h2 CaP (h2t) causes a prominent shift in subcellular localization from a more general actin-binding, to a strictly central stress-fiber-associated pattern. Neutralization of acidic residues by their uncharged counterparts (h2 ch-k.o.) has an effect on actin binding and localization similar to that seen following complete elimination of Thr tail sequences. Top row, actin visualized by Alexa 568 phalloidin; middle row, GFP fluorescence; bottom row, merged images.

View larger version (109K): [in a new window]   Fig. 5. Exchange of tail sequences in h1 CaP. Fusion of the tail sequences from either h2 CaP (h1-h2t) or ac. CaP(h1-act) alter the localization of the respective h1 CaP mutant construct in A7r5 cells and cause an increased association with peripheral actin structures, when compared with the wild-type molecule. Top row, actin visualized by Alexa 568 phalloidin; middle row, GFP fluorescence; bottom row, merged images.

View larger version (111K): [in a new window]   Fig. 6. Exchange of tail sequences in h2 CaP. Replacement of the h2 CaP tail with that of h1 (h2-h1t) CaP fails to regulate the actin-binding region. Fusion of either the h1 (h2-h1t) or the ac. CaP tail to h2 CaP (h2-act) only partially restores the autoinibition of the h2 tail. Top row, actin visualized by Alexa 568 phalloidin; middle row, GFP fluorescence; bottom row, merged images.

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