Limits...
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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Specificity of the [125I]α-actinin–CRP1 interaction. (A)  Coomassie blue–stained gel showing molecular mass markers and  the purified recombinant CRP1. Autoradiograph of parallel nitrocellulose strips probed with [125I]α-actinin in the absence of  competing protein (B), or in the presence of either a 2,000-fold  molar excess of unlabeled α-actinin (C), or a 2,000-fold molar excess of unlabeled BSA (D).
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Figure 3: Specificity of the [125I]α-actinin–CRP1 interaction. (A) Coomassie blue–stained gel showing molecular mass markers and the purified recombinant CRP1. Autoradiograph of parallel nitrocellulose strips probed with [125I]α-actinin in the absence of competing protein (B), or in the presence of either a 2,000-fold molar excess of unlabeled α-actinin (C), or a 2,000-fold molar excess of unlabeled BSA (D).

Mentions: To analyze further the specificity of the CRP1–α-actinin interaction, a competition experiment was performed using the blot overlay assay. Purified CRP1 was resolved by SDS-PAGE (Fig. 3 A) and transferred to nitrocellulose. The immobilized CRP1 was probed with radioiodinated α-actinin in the absence or presence of a 2,000-fold molar excess of unlabeled α-actinin or BSA (Fig. 3, B–D). The radioiodinated probe interacts with the purified CRP1 confirming that, under the conditions of this experiment, [125I]α-actinin interacts directly with CRP1. Moreover, in the presence of unlabeled α-actinin, but not in the presence of an equimolar amount of unlabeled BSA, the binding of the radioiodinated α-actinin to CRP1 was dramatically reduced. Collectively, these experiments demonstrate that in the blot overlay assay, the association between CRP1 and α-actinin is direct, specific and saturable.


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

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

Specificity of the [125I]α-actinin–CRP1 interaction. (A)  Coomassie blue–stained gel showing molecular mass markers and  the purified recombinant CRP1. Autoradiograph of parallel nitrocellulose strips probed with [125I]α-actinin in the absence of  competing protein (B), or in the presence of either a 2,000-fold  molar excess of unlabeled α-actinin (C), or a 2,000-fold molar excess of unlabeled BSA (D).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Specificity of the [125I]α-actinin–CRP1 interaction. (A) Coomassie blue–stained gel showing molecular mass markers and the purified recombinant CRP1. Autoradiograph of parallel nitrocellulose strips probed with [125I]α-actinin in the absence of competing protein (B), or in the presence of either a 2,000-fold molar excess of unlabeled α-actinin (C), or a 2,000-fold molar excess of unlabeled BSA (D).
Mentions: To analyze further the specificity of the CRP1–α-actinin interaction, a competition experiment was performed using the blot overlay assay. Purified CRP1 was resolved by SDS-PAGE (Fig. 3 A) and transferred to nitrocellulose. The immobilized CRP1 was probed with radioiodinated α-actinin in the absence or presence of a 2,000-fold molar excess of unlabeled α-actinin or BSA (Fig. 3, B–D). The radioiodinated probe interacts with the purified CRP1 confirming that, under the conditions of this experiment, [125I]α-actinin interacts directly with CRP1. Moreover, in the presence of unlabeled α-actinin, but not in the presence of an equimolar amount of unlabeled BSA, the binding of the radioiodinated α-actinin to CRP1 was dramatically reduced. Collectively, these experiments demonstrate that in the blot overlay assay, the association between CRP1 and α-actinin is direct, specific and saturable.

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