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CRP1, a LIM domain protein implicated in muscle differentiation, interacts with alpha-actinin.

Pomiès P, Louis HA, Beckerle MC - J. Cell Biol. (1997)

Bottom Line: The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells.Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton.The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.

ABSTRACT
Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that alpha-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

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Characterization of an anti-peptide antibody (B37) directed against cCRP1. (A) A Coomassie blue–stained gel showing molecular mass markers M and total CEF proteins L. (B) A  parallel gel was transferred to nitrocellulose and probed with the  anti-CRP1 antibody B37 or its corresponding preimmune serum  pre. A single polypeptide of 23 kD is recognized by the antibody.  (C) Autoradiograph of a gel loaded with a CEF lysate prepared  from [35S]methionine-cysteine–labeled cells L, the proteins immunoprecipitated from this lysate with the polyclonal antibody  raised against CRP1 B37, and with its corresponding preimmune  serum pre. A single protein of 23 kD is specifically immunoprecipitated with the antibody against CRP1.
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Figure 5: Characterization of an anti-peptide antibody (B37) directed against cCRP1. (A) A Coomassie blue–stained gel showing molecular mass markers M and total CEF proteins L. (B) A parallel gel was transferred to nitrocellulose and probed with the anti-CRP1 antibody B37 or its corresponding preimmune serum pre. A single polypeptide of 23 kD is recognized by the antibody. (C) Autoradiograph of a gel loaded with a CEF lysate prepared from [35S]methionine-cysteine–labeled cells L, the proteins immunoprecipitated from this lysate with the polyclonal antibody raised against CRP1 B37, and with its corresponding preimmune serum pre. A single protein of 23 kD is specifically immunoprecipitated with the antibody against CRP1.

Mentions: The work described above reports the ability of CRP1 and α-actinin to associate with each other in vitro. If this interaction also occurs in vivo, one might expect CRP1 and α-actinin to be colocalized in cells. To examine this possibility, we performed double-label immunofluorescence microscopy using an anti-peptide antibody (B37) raised against a sequence in cCRP1. By Western blot analysis of a CEF lysate, the B37 antibody recognizes a single band that exhibits an apparent molecular mass of 23 kD (Fig. 5 B) and comigrates with CRP1 (data not shown); no protein is detected using the preimmune serum (Fig. 5 B). Similarly, a single band that migrates at an apparent molecular mass of 23 kD is immunoprecipitated from a detergent extract of [35S]methionine–labeled CEF under denaturing conditions, whereas no immunoprecipitated band is detected under the same conditions when the preimmune serum is used (Fig. 5 C). The B37 antibody was used to compare the subcellular distributions of CRP1 and α-actinin using double-label indirect immunofluorescence in CEF cells and in a primary culture of smooth muscle cells from chicken gizzard (Fig. 6). By this approach, we observe that CRP1 and α-actinin are extensively colocalized in cells along the actin stress fibers (Fig. 6, C and F), in accordance with the idea that they could interact in vivo. We also observed that both proteins are present at the leading edge of the cells, and in the adhesion plaques (Fig. 6 F). However, in some adhesion plaques, where α-actinin is present, CRP1 is not detected (data not shown). This observation is consistent with a previous report showing that CRP1 is present in some adhesion plaques of CEF cells but not in others (Crawford et al., 1994).


CRP1, a LIM domain protein implicated in muscle differentiation, interacts with alpha-actinin.

Pomiès P, Louis HA, Beckerle MC - J. Cell Biol. (1997)

Characterization of an anti-peptide antibody (B37) directed against cCRP1. (A) A Coomassie blue–stained gel showing molecular mass markers M and total CEF proteins L. (B) A  parallel gel was transferred to nitrocellulose and probed with the  anti-CRP1 antibody B37 or its corresponding preimmune serum  pre. A single polypeptide of 23 kD is recognized by the antibody.  (C) Autoradiograph of a gel loaded with a CEF lysate prepared  from [35S]methionine-cysteine–labeled cells L, the proteins immunoprecipitated from this lysate with the polyclonal antibody  raised against CRP1 B37, and with its corresponding preimmune  serum pre. A single protein of 23 kD is specifically immunoprecipitated with the antibody against CRP1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139825&req=5

Figure 5: Characterization of an anti-peptide antibody (B37) directed against cCRP1. (A) A Coomassie blue–stained gel showing molecular mass markers M and total CEF proteins L. (B) A parallel gel was transferred to nitrocellulose and probed with the anti-CRP1 antibody B37 or its corresponding preimmune serum pre. A single polypeptide of 23 kD is recognized by the antibody. (C) Autoradiograph of a gel loaded with a CEF lysate prepared from [35S]methionine-cysteine–labeled cells L, the proteins immunoprecipitated from this lysate with the polyclonal antibody raised against CRP1 B37, and with its corresponding preimmune serum pre. A single protein of 23 kD is specifically immunoprecipitated with the antibody against CRP1.
Mentions: The work described above reports the ability of CRP1 and α-actinin to associate with each other in vitro. If this interaction also occurs in vivo, one might expect CRP1 and α-actinin to be colocalized in cells. To examine this possibility, we performed double-label immunofluorescence microscopy using an anti-peptide antibody (B37) raised against a sequence in cCRP1. By Western blot analysis of a CEF lysate, the B37 antibody recognizes a single band that exhibits an apparent molecular mass of 23 kD (Fig. 5 B) and comigrates with CRP1 (data not shown); no protein is detected using the preimmune serum (Fig. 5 B). Similarly, a single band that migrates at an apparent molecular mass of 23 kD is immunoprecipitated from a detergent extract of [35S]methionine–labeled CEF under denaturing conditions, whereas no immunoprecipitated band is detected under the same conditions when the preimmune serum is used (Fig. 5 C). The B37 antibody was used to compare the subcellular distributions of CRP1 and α-actinin using double-label indirect immunofluorescence in CEF cells and in a primary culture of smooth muscle cells from chicken gizzard (Fig. 6). By this approach, we observe that CRP1 and α-actinin are extensively colocalized in cells along the actin stress fibers (Fig. 6, C and F), in accordance with the idea that they could interact in vivo. We also observed that both proteins are present at the leading edge of the cells, and in the adhesion plaques (Fig. 6 F). However, in some adhesion plaques, where α-actinin is present, CRP1 is not detected (data not shown). This observation is consistent with a previous report showing that CRP1 is present in some adhesion plaques of CEF cells but not in others (Crawford et al., 1994).

Bottom Line: The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells.Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton.The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.

ABSTRACT
Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that alpha-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

Show MeSH
Related in: MedlinePlus