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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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An in vivo interaction between CRP1 and  α-actinin in smooth muscle  cells. Proteins were immunoprecipitated from a chicken  gizzard smooth muscle lysate L with the polyclonal antibody raised against CRP1  B37 and with the corresponding preimmune serum pre.  The immunoprecipitated proteins were resolved by SDS-PAGE and were transferred  to nitrocellulose and probed  with polyclonal antibodies  raised against CRP1 (A) or  α-actinin (B). α-actinin is immunoprecipitated under nondenaturing conditions with the anti-CRP1 antibody, but not with the preimmune serum. The position  of the molecular mass markers is indicated on the left in kD.
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Figure 7: An in vivo interaction between CRP1 and α-actinin in smooth muscle cells. Proteins were immunoprecipitated from a chicken gizzard smooth muscle lysate L with the polyclonal antibody raised against CRP1 B37 and with the corresponding preimmune serum pre. The immunoprecipitated proteins were resolved by SDS-PAGE and were transferred to nitrocellulose and probed with polyclonal antibodies raised against CRP1 (A) or α-actinin (B). α-actinin is immunoprecipitated under nondenaturing conditions with the anti-CRP1 antibody, but not with the preimmune serum. The position of the molecular mass markers is indicated on the left in kD.

Mentions: We performed a coimmunoprecipitation experiment to evaluate the ability of CRP1 to interact with α-actinin in vivo. CRP1 can be immunoprecipitated from a smooth muscle cell extract of smooth muscle cells under nondenaturing conditions using the B37 anti–CRP1 antibody (Fig. 7 A). Under these conditions, α-actinin is detected in the immunoprecipitate with CRP1 (Fig. 7 B), whereas another cytoskeletal protein, vinculin, is not detected (data not shown). Neither CRP1 nor α-actinin is detected when the preimmune serum is used in the immunoprecipitation assay (Fig. 7, A and B). These data provide evidence that α-actinin and CRP1 can be recovered as a complex from smooth muscle cells.


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

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

An in vivo interaction between CRP1 and  α-actinin in smooth muscle  cells. Proteins were immunoprecipitated from a chicken  gizzard smooth muscle lysate L with the polyclonal antibody raised against CRP1  B37 and with the corresponding preimmune serum pre.  The immunoprecipitated proteins were resolved by SDS-PAGE and were transferred  to nitrocellulose and probed  with polyclonal antibodies  raised against CRP1 (A) or  α-actinin (B). α-actinin is immunoprecipitated under nondenaturing conditions with the anti-CRP1 antibody, but not with the preimmune serum. 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 7: An in vivo interaction between CRP1 and α-actinin in smooth muscle cells. Proteins were immunoprecipitated from a chicken gizzard smooth muscle lysate L with the polyclonal antibody raised against CRP1 B37 and with the corresponding preimmune serum pre. The immunoprecipitated proteins were resolved by SDS-PAGE and were transferred to nitrocellulose and probed with polyclonal antibodies raised against CRP1 (A) or α-actinin (B). α-actinin is immunoprecipitated under nondenaturing conditions with the anti-CRP1 antibody, but not with the preimmune serum. The position of the molecular mass markers is indicated on the left in kD.
Mentions: We performed a coimmunoprecipitation experiment to evaluate the ability of CRP1 to interact with α-actinin in vivo. CRP1 can be immunoprecipitated from a smooth muscle cell extract of smooth muscle cells under nondenaturing conditions using the B37 anti–CRP1 antibody (Fig. 7 A). Under these conditions, α-actinin is detected in the immunoprecipitate with CRP1 (Fig. 7 B), whereas another cytoskeletal protein, vinculin, is not detected (data not shown). Neither CRP1 nor α-actinin is detected when the preimmune serum is used in the immunoprecipitation assay (Fig. 7, A and B). These data provide evidence that α-actinin and CRP1 can be recovered as a complex from smooth muscle cells.

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