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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The binding site for α-actinin on CRP1 is contained  within the CRP1-LIM1 fragment. (A) Coomassie blue–stained  gel showing the purified CRP1 (lane 1), the purified CRP1-LIM1  fragment (lane 2) and the purified CRP1-LIM2 fragment (lane  3). 100 pmoles of CRP1, 200 pmoles of CRP1-LIM1, and 200  pmoles of CRP1-LIM2 were loaded on the gel. The positions of  CRP1, CRP1-LIM1, and CRP1-LIM2 are marked (CRP1, LIM1,  and LIM2, respectively). The corresponding blot overlay assay  probed with [125I]α-actinin is shown in B. (C) Autoradiograph illustrating 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 9: The binding site for α-actinin on CRP1 is contained within the CRP1-LIM1 fragment. (A) Coomassie blue–stained gel showing the purified CRP1 (lane 1), the purified CRP1-LIM1 fragment (lane 2) and the purified CRP1-LIM2 fragment (lane 3). 100 pmoles of CRP1, 200 pmoles of CRP1-LIM1, and 200 pmoles of CRP1-LIM2 were loaded on the gel. The positions of CRP1, CRP1-LIM1, and CRP1-LIM2 are marked (CRP1, LIM1, and LIM2, respectively). The corresponding blot overlay assay probed with [125I]α-actinin is shown in B. (C) Autoradiograph illustrating the purity of the radioiodinated α-actinin probe. The position of the molecular mass markers is indicated on the left in kD.

Mentions: CRP1 displays two LIM domains separated by 56 amino acids (Crawford et al., 1994). To characterize the binding site for α-actinin on CRP1, we compared the ability of α-actinin to interact with full-length CRP1 and two peptides, CRP1-LIM1 and CRP1-LIM2, derived from the intact protein. CRP1-LIM1 corresponds to the NH2-terminal part of CRP1 (aa 1–107) containing the NH2-terminal LIM domain followed by the first glycine-rich repeat of the protein, and CRP1-LIM2 corresponds to the COOH-terminal part of the protein (aa 108–192) containing the COOH-terminal LIM domain and the second glycine-rich repeat. CRP1, CRP1-LIM1, and CRP1-LIM2 were resolved by SDS-PAGE (Fig. 9 A), were transferred to nitrocellulose and were probed for their ability to interact with [125I]α-actinin in a blot overlay assay (Fig. 9 B). The radioiodinated α-actinin interacts with the bacterially expressed purified CRP1 and with CRP1-LIM1. The molar amounts of the two single LIM peptides, CRP1-LIM1 and CRP1-LIM2, loaded on the gel was twice the amount loaded for the double LIM protein, CRP1. Although the [125I]α-actinin bound only to intact CRP1 and the CRP1-LIM1 peptide, the binding to the deletion construct reached only about 50% of the binding observed with full-length CRP1, as measured by PhosphorImager analysis (data not shown). No interaction is detected between the [125I]α-actinin and CRP1-LIM2. Thus it appears that the CRP1-LIM1 peptide contains sequence information that establishes a docking site for α-actinin; the generation of the CRP-LIM1 truncation may have rendered the α-actinin binding site suboptimal. We cannot rule out the possibility that other low affinity binding sites for α-actinin exist in CRP1, however, the only site we have been able to map is within the CRP1-LIM1 region.


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

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

The binding site for α-actinin on CRP1 is contained  within the CRP1-LIM1 fragment. (A) Coomassie blue–stained  gel showing the purified CRP1 (lane 1), the purified CRP1-LIM1  fragment (lane 2) and the purified CRP1-LIM2 fragment (lane  3). 100 pmoles of CRP1, 200 pmoles of CRP1-LIM1, and 200  pmoles of CRP1-LIM2 were loaded on the gel. The positions of  CRP1, CRP1-LIM1, and CRP1-LIM2 are marked (CRP1, LIM1,  and LIM2, respectively). The corresponding blot overlay assay  probed with [125I]α-actinin is shown in B. (C) Autoradiograph illustrating 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 9: The binding site for α-actinin on CRP1 is contained within the CRP1-LIM1 fragment. (A) Coomassie blue–stained gel showing the purified CRP1 (lane 1), the purified CRP1-LIM1 fragment (lane 2) and the purified CRP1-LIM2 fragment (lane 3). 100 pmoles of CRP1, 200 pmoles of CRP1-LIM1, and 200 pmoles of CRP1-LIM2 were loaded on the gel. The positions of CRP1, CRP1-LIM1, and CRP1-LIM2 are marked (CRP1, LIM1, and LIM2, respectively). The corresponding blot overlay assay probed with [125I]α-actinin is shown in B. (C) Autoradiograph illustrating the purity of the radioiodinated α-actinin probe. The position of the molecular mass markers is indicated on the left in kD.
Mentions: CRP1 displays two LIM domains separated by 56 amino acids (Crawford et al., 1994). To characterize the binding site for α-actinin on CRP1, we compared the ability of α-actinin to interact with full-length CRP1 and two peptides, CRP1-LIM1 and CRP1-LIM2, derived from the intact protein. CRP1-LIM1 corresponds to the NH2-terminal part of CRP1 (aa 1–107) containing the NH2-terminal LIM domain followed by the first glycine-rich repeat of the protein, and CRP1-LIM2 corresponds to the COOH-terminal part of the protein (aa 108–192) containing the COOH-terminal LIM domain and the second glycine-rich repeat. CRP1, CRP1-LIM1, and CRP1-LIM2 were resolved by SDS-PAGE (Fig. 9 A), were transferred to nitrocellulose and were probed for their ability to interact with [125I]α-actinin in a blot overlay assay (Fig. 9 B). The radioiodinated α-actinin interacts with the bacterially expressed purified CRP1 and with CRP1-LIM1. The molar amounts of the two single LIM peptides, CRP1-LIM1 and CRP1-LIM2, loaded on the gel was twice the amount loaded for the double LIM protein, CRP1. Although the [125I]α-actinin bound only to intact CRP1 and the CRP1-LIM1 peptide, the binding to the deletion construct reached only about 50% of the binding observed with full-length CRP1, as measured by PhosphorImager analysis (data not shown). No interaction is detected between the [125I]α-actinin and CRP1-LIM2. Thus it appears that the CRP1-LIM1 peptide contains sequence information that establishes a docking site for α-actinin; the generation of the CRP-LIM1 truncation may have rendered the α-actinin binding site suboptimal. We cannot rule out the possibility that other low affinity binding sites for α-actinin exist in CRP1, however, the only site we have been able to map is within the CRP1-LIM1 region.

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