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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Quantitation of abLIM binding to actin filaments. Assay mixtures containing different total amounts of GST–abLIM  polypeptide (putative actin-binding domain of abLIM, amino acids 585–778) were processed to separate bound abLIM in each  pellet fraction (P) from free abLIM in the supernatant fractions  (S). The derived values are plotted in a. The total amount of  abLIM added (μg) in each 60-μl reaction mixture is indicated  above each pair of gel lanes in b.
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Figure 7: Quantitation of abLIM binding to actin filaments. Assay mixtures containing different total amounts of GST–abLIM polypeptide (putative actin-binding domain of abLIM, amino acids 585–778) were processed to separate bound abLIM in each pellet fraction (P) from free abLIM in the supernatant fractions (S). The derived values are plotted in a. The total amount of abLIM added (μg) in each 60-μl reaction mixture is indicated above each pair of gel lanes in b.

Mentions: To date, it has not been possible to express the full-length abLIM sequence in vitro as a GST fusion protein. However, the actin-binding affinity and stoichiometry of the shorter expressed GST–dem construct were estimated by quantitative application of the sedimentation assay described above (Fig. 7). The calculated dissociation constant for the abLIM/F–actin interaction is ∼2.6 μM, assuming a single binding site and a molecular mass for abLIM equal to the abLIM (amino acids 585–778) plus the GST polypeptide. The maximal binding stoichiometry of abLIM is approximately one GST–abLIM monomer per 1.1 actin molecules.


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)

Quantitation of abLIM binding to actin filaments. Assay mixtures containing different total amounts of GST–abLIM  polypeptide (putative actin-binding domain of abLIM, amino acids 585–778) were processed to separate bound abLIM in each  pellet fraction (P) from free abLIM in the supernatant fractions  (S). The derived values are plotted in a. The total amount of  abLIM added (μg) in each 60-μl reaction mixture is indicated  above each pair of gel lanes in b.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Quantitation of abLIM binding to actin filaments. Assay mixtures containing different total amounts of GST–abLIM polypeptide (putative actin-binding domain of abLIM, amino acids 585–778) were processed to separate bound abLIM in each pellet fraction (P) from free abLIM in the supernatant fractions (S). The derived values are plotted in a. The total amount of abLIM added (μg) in each 60-μl reaction mixture is indicated above each pair of gel lanes in b.
Mentions: To date, it has not been possible to express the full-length abLIM sequence in vitro as a GST fusion protein. However, the actin-binding affinity and stoichiometry of the shorter expressed GST–dem construct were estimated by quantitative application of the sedimentation assay described above (Fig. 7). The calculated dissociation constant for the abLIM/F–actin interaction is ∼2.6 μM, assuming a single binding site and a molecular mass for abLIM equal to the abLIM (amino acids 585–778) plus the GST polypeptide. The maximal binding stoichiometry of abLIM is approximately one GST–abLIM monomer per 1.1 actin molecules.

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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