Limits...
Molecular characterization of abLIM, a novel actin-binding and double zinc finger protein.

Roof DJ, Hayes A, Adamian M, Chishti AH, Li T - J. Cell Biol. (1997)

Bottom Line: However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments.Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners.The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.

View Article: PubMed Central - PubMed

Affiliation: Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.

ABSTRACT
Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is approximately 50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.

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Actin binding to three abLIM isoforms expressed in  vitro. Polypeptides expressed in vitro using constructs corresponding to abLIM-l, abLIM-m, and abLIM-s were tested for  their ability to bind to actin filaments. All three isoforms cosediment with F-actin (+actin) and are found primarily in the respective pellet fractions (P). Each isoform is found in the supernatant  fraction (S) when actin is not present in the assay mixture  (−actin). Nonspecific binding to the GST-only polypeptide was  negligible at all concentrations (data not shown). Approximate  Mr (kD) of each isoform is indicated at left.
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Figure 5: Actin binding to three abLIM isoforms expressed in vitro. Polypeptides expressed in vitro using constructs corresponding to abLIM-l, abLIM-m, and abLIM-s were tested for their ability to bind to actin filaments. All three isoforms cosediment with F-actin (+actin) and are found primarily in the respective pellet fractions (P). Each isoform is found in the supernatant fraction (S) when actin is not present in the assay mixture (−actin). Nonspecific binding to the GST-only polypeptide was negligible at all concentrations (data not shown). Approximate Mr (kD) of each isoform is indicated at left.

Mentions: The function of abLIM protein, particularly within some restricted subcellular locales, may be related to its ability to bind to actin filaments. This property is strongly predicted from abLIM sequence homology with the known actin-binding proteins, erythrocyte dematin and villin. To verify that abLIM indeed binds actin, all three abLIM isoforms were expressed in vitro using a reticulocyte lysate system, and the actin-binding ability of each expressed isoform was tested independently (Fig. 5). In each case, the presence of actin in the reaction mixture shifted the expressed polypeptides from the supernatant to the pellet fraction. This indicates that all three abLIM isoforms have the capacity to bind to actin filaments in vitro.


Molecular characterization of abLIM, a novel actin-binding and double zinc finger protein.

Roof DJ, Hayes A, Adamian M, Chishti AH, Li T - J. Cell Biol. (1997)

Actin binding to three abLIM isoforms expressed in  vitro. Polypeptides expressed in vitro using constructs corresponding to abLIM-l, abLIM-m, and abLIM-s were tested for  their ability to bind to actin filaments. All three isoforms cosediment with F-actin (+actin) and are found primarily in the respective pellet fractions (P). Each isoform is found in the supernatant  fraction (S) when actin is not present in the assay mixture  (−actin). Nonspecific binding to the GST-only polypeptide was  negligible at all concentrations (data not shown). Approximate  Mr (kD) of each isoform is indicated at left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141644&req=5

Figure 5: Actin binding to three abLIM isoforms expressed in vitro. Polypeptides expressed in vitro using constructs corresponding to abLIM-l, abLIM-m, and abLIM-s were tested for their ability to bind to actin filaments. All three isoforms cosediment with F-actin (+actin) and are found primarily in the respective pellet fractions (P). Each isoform is found in the supernatant fraction (S) when actin is not present in the assay mixture (−actin). Nonspecific binding to the GST-only polypeptide was negligible at all concentrations (data not shown). Approximate Mr (kD) of each isoform is indicated at left.
Mentions: The function of abLIM protein, particularly within some restricted subcellular locales, may be related to its ability to bind to actin filaments. This property is strongly predicted from abLIM sequence homology with the known actin-binding proteins, erythrocyte dematin and villin. To verify that abLIM indeed binds actin, all three abLIM isoforms were expressed in vitro using a reticulocyte lysate system, and the actin-binding ability of each expressed isoform was tested independently (Fig. 5). In each case, the presence of actin in the reaction mixture shifted the expressed polypeptides from the supernatant to the pellet fraction. This indicates that all three abLIM isoforms have the capacity to bind to actin filaments in vitro.

Bottom Line: However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments.Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners.The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.

View Article: PubMed Central - PubMed

Affiliation: Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.

ABSTRACT
Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is approximately 50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.

Show MeSH