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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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Demonstration of a direct interaction between CRP1  and [125I]α-actinin using a blot overlay assay. (A) Coomassie  blue–stained gel showing a 27–34 (lane 1), a 34–43 (lane 2), and a  43–61% (lane 3) ammonium sulfate precipitates from an avian  smooth muscle extract. Proteins from a parallel gel were transferred to nitrocellulose and the nitrocellulose strip was probed  with [125I]α-actinin. The resulting autoradiograph shown in B illustrates [125I]α-actinin binding to CRP1. (C) Autoradiograph  demonstrating the purity of the radioiodinated α-actinin probe.  The position of the molecular mass markers is indicated on the  left, in kD.
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Figure 2: Demonstration of a direct interaction between CRP1 and [125I]α-actinin using a blot overlay assay. (A) Coomassie blue–stained gel showing a 27–34 (lane 1), a 34–43 (lane 2), and a 43–61% (lane 3) ammonium sulfate precipitates from an avian smooth muscle extract. Proteins from a parallel gel were transferred to nitrocellulose and the nitrocellulose strip was probed with [125I]α-actinin. The resulting autoradiograph shown in B illustrates [125I]α-actinin binding to CRP1. (C) Autoradiograph demonstrating the purity of the radioiodinated α-actinin probe. The position of the molecular mass markers is indicated on the left, in kD.

Mentions: We have further characterized the CRP1–α-actinin interaction using a blot overlay assay that has been used previously to study many protein–protein interactions (Belkin and Koteliansky, 1987; Crawford et al., 1992; Sadler et al., 1992). We evaluated the ability of [125I]α-actinin to bind directly to CRP1 present in fractions derived from an avian smooth muscle extract. Three different ammonium sulfate precipitates that include a diverse collection of smooth muscle-derived proteins were resolved by SDS-PAGE (Fig. 2 A) and transferred to nitrocellulose. CRP1 is found in the 34–43% ammonium sulfate (Fig. 2, lane 2) but not in the 27–34 and the 43–61% ammonium sulfate precipitates. Purified α-actinin was radioiodinated and used as a probe to examine its ability to interact with CRP1 that was immobilized on nitrocellulose (Fig. 2 B). The purity of the [125I]α-actinin used in this experiment is shown in Fig. 2 C. Among the proteins that are precipitated from the smooth muscle extract, [125I]α-actinin recognizes a protein that exhibits an apparent molecular mass of 23 kD, corresponding to the molecular mass of CRP1. A number of other abundant proteins present on the nitrocellulose membrane fail to interact with the radioiodinated α-actinin showing the selectivity of the radiolabeled probe.


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

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

Demonstration of a direct interaction between CRP1  and [125I]α-actinin using a blot overlay assay. (A) Coomassie  blue–stained gel showing a 27–34 (lane 1), a 34–43 (lane 2), and a  43–61% (lane 3) ammonium sulfate precipitates from an avian  smooth muscle extract. Proteins from a parallel gel were transferred to nitrocellulose and the nitrocellulose strip was probed  with [125I]α-actinin. The resulting autoradiograph shown in B illustrates [125I]α-actinin binding to CRP1. (C) Autoradiograph  demonstrating the purity of the radioiodinated α-actinin probe.  The position of the molecular mass markers is indicated on the  left, in kD.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Demonstration of a direct interaction between CRP1 and [125I]α-actinin using a blot overlay assay. (A) Coomassie blue–stained gel showing a 27–34 (lane 1), a 34–43 (lane 2), and a 43–61% (lane 3) ammonium sulfate precipitates from an avian smooth muscle extract. Proteins from a parallel gel were transferred to nitrocellulose and the nitrocellulose strip was probed with [125I]α-actinin. The resulting autoradiograph shown in B illustrates [125I]α-actinin binding to CRP1. (C) Autoradiograph demonstrating the purity of the radioiodinated α-actinin probe. The position of the molecular mass markers is indicated on the left, in kD.
Mentions: We have further characterized the CRP1–α-actinin interaction using a blot overlay assay that has been used previously to study many protein–protein interactions (Belkin and Koteliansky, 1987; Crawford et al., 1992; Sadler et al., 1992). We evaluated the ability of [125I]α-actinin to bind directly to CRP1 present in fractions derived from an avian smooth muscle extract. Three different ammonium sulfate precipitates that include a diverse collection of smooth muscle-derived proteins were resolved by SDS-PAGE (Fig. 2 A) and transferred to nitrocellulose. CRP1 is found in the 34–43% ammonium sulfate (Fig. 2, lane 2) but not in the 27–34 and the 43–61% ammonium sulfate precipitates. Purified α-actinin was radioiodinated and used as a probe to examine its ability to interact with CRP1 that was immobilized on nitrocellulose (Fig. 2 B). The purity of the [125I]α-actinin used in this experiment is shown in Fig. 2 C. Among the proteins that are precipitated from the smooth muscle extract, [125I]α-actinin recognizes a protein that exhibits an apparent molecular mass of 23 kD, corresponding to the molecular mass of CRP1. A number of other abundant proteins present on the nitrocellulose membrane fail to interact with the radioiodinated α-actinin showing the selectivity of the radiolabeled probe.

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