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.

Show MeSH

Related in: MedlinePlus

Specificity of the α-actinin–CRP1 interaction under  nondenaturing conditions. (A) A Coomassie blue–stained gel  showing molecular mass markers M, purified α-actinin (lane 1),  and the 27–34% ammonium sulfate precipitate from avian  smooth muscle extract (lane 2) that was loaded onto the affinity  columns and used in the affinity resin binding assay. (B) Lane 1,  Western immunoblot analysis of the 27–34% ammonium sulfate  precipitate that was loaded onto the affinity columns using a  polyclonal antibody raised against chicken α-actinin; lane 2, silver-stained gel showing the proteins eluted from the CRP1 column; lane 3, Western immunoblot analysis of the proteins shown  in lane 2 using a polyclonal antibody raised against α-actinin; lane 4,  silver-stained gel showing the material eluted from the BSA column; lane 5, Western immunoblot revealed that no α-actinin  bound to the BSA column (α-a, α-actinin). (C) Coomassie blue– stained gel showing the purified GST (lane 1) and GST-CRP1  (lane 2) proteins that were used to generate the affinity resins.  (D) Western immunoblot analysis to detect chicken α-actinin.  The gel was loaded with α-actinin (lane 1) or a 27–34% ammonium sulfate precipitate from a smooth muscle cell extract (lane  2). Purified α-actinin or proteins found in the 27–34% ammonium sulfate precipitate were incubated with GST agarose (lanes  3 and 5) or GST-CRP1 agarose (lanes 4 and 6). α-Actinin binds  to the GST-CRP1 affinity resin. A mock affinity resin binding assay was performed with GST-CRP1 agarose beads in the absence  of α-actinin; no immunoreactive product is observed (lane 7). (E)  [125I]α-actinin was incubated with GST-CRP1 (left) or GST agarose beads (right) in the absence of competing proteins (+  buffer), in the presence of a 2,000-fold molar excess of unlabeled  α-actinin (+ unlabeled α-actinin), or in the presence of an equivalent molar amount of BSA (+ BSA). The counts bound to the  agarose beads were analyzed using a γ counter and expressed as a  percentage of bound [125I]α-actinin in absence of competing proteins. Mean and SEM from three experiments are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139825&req=5

Figure 1: Specificity of the α-actinin–CRP1 interaction under nondenaturing conditions. (A) A Coomassie blue–stained gel showing molecular mass markers M, purified α-actinin (lane 1), and the 27–34% ammonium sulfate precipitate from avian smooth muscle extract (lane 2) that was loaded onto the affinity columns and used in the affinity resin binding assay. (B) Lane 1, Western immunoblot analysis of the 27–34% ammonium sulfate precipitate that was loaded onto the affinity columns using a polyclonal antibody raised against chicken α-actinin; lane 2, silver-stained gel showing the proteins eluted from the CRP1 column; lane 3, Western immunoblot analysis of the proteins shown in lane 2 using a polyclonal antibody raised against α-actinin; lane 4, silver-stained gel showing the material eluted from the BSA column; lane 5, Western immunoblot revealed that no α-actinin bound to the BSA column (α-a, α-actinin). (C) Coomassie blue– stained gel showing the purified GST (lane 1) and GST-CRP1 (lane 2) proteins that were used to generate the affinity resins. (D) Western immunoblot analysis to detect chicken α-actinin. The gel was loaded with α-actinin (lane 1) or a 27–34% ammonium sulfate precipitate from a smooth muscle cell extract (lane 2). Purified α-actinin or proteins found in the 27–34% ammonium sulfate precipitate were incubated with GST agarose (lanes 3 and 5) or GST-CRP1 agarose (lanes 4 and 6). α-Actinin binds to the GST-CRP1 affinity resin. A mock affinity resin binding assay was performed with GST-CRP1 agarose beads in the absence of α-actinin; no immunoreactive product is observed (lane 7). (E) [125I]α-actinin was incubated with GST-CRP1 (left) or GST agarose beads (right) in the absence of competing proteins (+ buffer), in the presence of a 2,000-fold molar excess of unlabeled α-actinin (+ unlabeled α-actinin), or in the presence of an equivalent molar amount of BSA (+ BSA). The counts bound to the agarose beads were analyzed using a γ counter and expressed as a percentage of bound [125I]α-actinin in absence of competing proteins. Mean and SEM from three experiments are shown.

Mentions: Affinity chromatography was used to identify CRP1-binding proteins in an avian smooth muscle extract. Briefly, proteins extracted from smooth muscle preparations were fractionated by precipitation with increasing amounts of ammonium sulfate (27–34, 34–43, and 43–61% saturation). Each of the ammonium sulfate precipitates was subjected to affinity chromatography on a CRP1 column. The CRP1 column was prepared from bacterially expressed avian smooth muscle CRP1. We have shown previously that bacterially expressed CRP1 exhibits a native structure (Michelsen et al., 1993, 1994) and retains the ability to bind zyxin (Schmeichel and Beckerle, 1994). When a 27–34% ammonium sulfate precipitate from the avian smooth muscle extract (Fig. 1 A, lane 2) is loaded on a CRP1 column, four proteins of ∼115, 100, 41, and 35 kD elute from the column with a high salt buffer as detected by silver staining (Fig. 1 B, lane 2). By Western immunoblot analysis using specific antibodies, we determined that the 100-kD protein that binds to the CRP1 column is α-actinin (Fig. 1 B, lane 3). No protein was detected using antibodies against the two cytoskeletal proteins, talin and vinculin (data not shown). Because zyxin has previously been shown to interact with both α-actinin and CRP1 (Crawford et al., 1992; Sadler et al., 1992), zyxin could theoretically have been responsible for linking α-actinin to CRP1 in this experiment. However, no zyxin is detected in the 27–34% ammonium sulfate precipitate (Crawford and Beckerle, 1991), and therefore the CRP1-α-actinin interaction can not be mediated by zyxin. In control experiments, the 27–34% ammonium sulfate precipitate was loaded on a BSA column. In this case, no α-actinin was recovered after a high salt buffer elution as monitored by silver staining and Western immunoblot (Fig. 1 B, lanes 4 and 5). Collectively, the results of these experiments suggest that CRP1 can interact either directly or indirectly with the actin binding protein α-actinin.


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 α-actinin–CRP1 interaction under  nondenaturing conditions. (A) A Coomassie blue–stained gel  showing molecular mass markers M, purified α-actinin (lane 1),  and the 27–34% ammonium sulfate precipitate from avian  smooth muscle extract (lane 2) that was loaded onto the affinity  columns and used in the affinity resin binding assay. (B) Lane 1,  Western immunoblot analysis of the 27–34% ammonium sulfate  precipitate that was loaded onto the affinity columns using a  polyclonal antibody raised against chicken α-actinin; lane 2, silver-stained gel showing the proteins eluted from the CRP1 column; lane 3, Western immunoblot analysis of the proteins shown  in lane 2 using a polyclonal antibody raised against α-actinin; lane 4,  silver-stained gel showing the material eluted from the BSA column; lane 5, Western immunoblot revealed that no α-actinin  bound to the BSA column (α-a, α-actinin). (C) Coomassie blue– stained gel showing the purified GST (lane 1) and GST-CRP1  (lane 2) proteins that were used to generate the affinity resins.  (D) Western immunoblot analysis to detect chicken α-actinin.  The gel was loaded with α-actinin (lane 1) or a 27–34% ammonium sulfate precipitate from a smooth muscle cell extract (lane  2). Purified α-actinin or proteins found in the 27–34% ammonium sulfate precipitate were incubated with GST agarose (lanes  3 and 5) or GST-CRP1 agarose (lanes 4 and 6). α-Actinin binds  to the GST-CRP1 affinity resin. A mock affinity resin binding assay was performed with GST-CRP1 agarose beads in the absence  of α-actinin; no immunoreactive product is observed (lane 7). (E)  [125I]α-actinin was incubated with GST-CRP1 (left) or GST agarose beads (right) in the absence of competing proteins (+  buffer), in the presence of a 2,000-fold molar excess of unlabeled  α-actinin (+ unlabeled α-actinin), or in the presence of an equivalent molar amount of BSA (+ BSA). The counts bound to the  agarose beads were analyzed using a γ counter and expressed as a  percentage of bound [125I]α-actinin in absence of competing proteins. Mean and SEM from three experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Specificity of the α-actinin–CRP1 interaction under nondenaturing conditions. (A) A Coomassie blue–stained gel showing molecular mass markers M, purified α-actinin (lane 1), and the 27–34% ammonium sulfate precipitate from avian smooth muscle extract (lane 2) that was loaded onto the affinity columns and used in the affinity resin binding assay. (B) Lane 1, Western immunoblot analysis of the 27–34% ammonium sulfate precipitate that was loaded onto the affinity columns using a polyclonal antibody raised against chicken α-actinin; lane 2, silver-stained gel showing the proteins eluted from the CRP1 column; lane 3, Western immunoblot analysis of the proteins shown in lane 2 using a polyclonal antibody raised against α-actinin; lane 4, silver-stained gel showing the material eluted from the BSA column; lane 5, Western immunoblot revealed that no α-actinin bound to the BSA column (α-a, α-actinin). (C) Coomassie blue– stained gel showing the purified GST (lane 1) and GST-CRP1 (lane 2) proteins that were used to generate the affinity resins. (D) Western immunoblot analysis to detect chicken α-actinin. The gel was loaded with α-actinin (lane 1) or a 27–34% ammonium sulfate precipitate from a smooth muscle cell extract (lane 2). Purified α-actinin or proteins found in the 27–34% ammonium sulfate precipitate were incubated with GST agarose (lanes 3 and 5) or GST-CRP1 agarose (lanes 4 and 6). α-Actinin binds to the GST-CRP1 affinity resin. A mock affinity resin binding assay was performed with GST-CRP1 agarose beads in the absence of α-actinin; no immunoreactive product is observed (lane 7). (E) [125I]α-actinin was incubated with GST-CRP1 (left) or GST agarose beads (right) in the absence of competing proteins (+ buffer), in the presence of a 2,000-fold molar excess of unlabeled α-actinin (+ unlabeled α-actinin), or in the presence of an equivalent molar amount of BSA (+ BSA). The counts bound to the agarose beads were analyzed using a γ counter and expressed as a percentage of bound [125I]α-actinin in absence of competing proteins. Mean and SEM from three experiments are shown.
Mentions: Affinity chromatography was used to identify CRP1-binding proteins in an avian smooth muscle extract. Briefly, proteins extracted from smooth muscle preparations were fractionated by precipitation with increasing amounts of ammonium sulfate (27–34, 34–43, and 43–61% saturation). Each of the ammonium sulfate precipitates was subjected to affinity chromatography on a CRP1 column. The CRP1 column was prepared from bacterially expressed avian smooth muscle CRP1. We have shown previously that bacterially expressed CRP1 exhibits a native structure (Michelsen et al., 1993, 1994) and retains the ability to bind zyxin (Schmeichel and Beckerle, 1994). When a 27–34% ammonium sulfate precipitate from the avian smooth muscle extract (Fig. 1 A, lane 2) is loaded on a CRP1 column, four proteins of ∼115, 100, 41, and 35 kD elute from the column with a high salt buffer as detected by silver staining (Fig. 1 B, lane 2). By Western immunoblot analysis using specific antibodies, we determined that the 100-kD protein that binds to the CRP1 column is α-actinin (Fig. 1 B, lane 3). No protein was detected using antibodies against the two cytoskeletal proteins, talin and vinculin (data not shown). Because zyxin has previously been shown to interact with both α-actinin and CRP1 (Crawford et al., 1992; Sadler et al., 1992), zyxin could theoretically have been responsible for linking α-actinin to CRP1 in this experiment. However, no zyxin is detected in the 27–34% ammonium sulfate precipitate (Crawford and Beckerle, 1991), and therefore the CRP1-α-actinin interaction can not be mediated by zyxin. In control experiments, the 27–34% ammonium sulfate precipitate was loaded on a BSA column. In this case, no α-actinin was recovered after a high salt buffer elution as monitored by silver staining and Western immunoblot (Fig. 1 B, lanes 4 and 5). Collectively, the results of these experiments suggest that CRP1 can interact either directly or indirectly with the actin binding protein α-actinin.

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