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A conserved sequence in calmodulin regulated spectrin-associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth.

King MD, Phillips GW, Bignone PA, Hayes NV, Pinder JC, Baines AJ - J. Neurochem. (2013)

Bottom Line: Calmodulin regulated spectrin-associated protein 1 (CAMSAP1) is a vertebrate microtubule-binding protein, and a representative of a family of cytoskeletal proteins that arose with animals.Biol.We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin-binding to neurite outgrowth.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Kent, Kent, UK.

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Related in: MedlinePlus

Interaction of calmodulin regulated spectrin-associated protein 1 (CAMSAP1) with spectrin. (a) Generalized structures of spectrin and C-terminal variants of β-spectrins. Spectrin α and β subunits are arranged in tetramers. At the C-terminal end of β chains, splice variation gives rise to long (βIIΣI) and short (βIIΣ2) variants. The long variant has a PH domain joined to the last of the helical repeats (repeats 16 and 17) by a linker of around 90 amino acids. Differential mRNA splicing gives rise to a variant in which this linker is interrupted such that the PH domain is lost and the second half of the linker is replaced with a unique (probably unstructured) region in the short variant. (b) Affinity chromatography of CAMSAP1 on various spectrin constructs coupled to Sepharose. A his-tagged central region construct (see Fig 1a) was flowed over the indicated spectrin protein or fragment coupled to Sepharose. Unbound material was washed away, and bound material recovered by elution with 1 M KI. Fractions representing bound material were analysed by immunoblot: sequential fractions from the 1M KI elution, labelled 1–4, are shown here. Note that the CAMSAP1 fragment bound to whole brain spectrin, the βIIΣI C-terminal fragment from repeat 16 to the C-terminus, the βIIΣI linker fragment, but not to the fragment of βIIΣ2 repeat 16 to C-terminus or the PH domain alone. (c) Interaction of brain spectrin with the CAMSAP-conserved region 1 (CC1) region. His-tagged CC1 construct was coupled to Sepaharose and mixed with brain extract. The figure shows an immunoblot of material recovered after washing the beads in buffer and elution with sodium dodecyl sulfate (SDS). Lanes 1-4 show immunoblots probed with antibody to brain spectrin. Lane 1: CC1-Sepharose. Lane 2: bovine serum albumin coupled to Sepharose. Lane 3: Unconjugated Sepharose. Lane 4: AAAA mutant. (d) Interaction of peptides from CC1 with spectrin. Biotinylated peptides CC1-P1-4 (see Methods and Fig 1b) were mixed with brain extract and recovered on streptavidin-Sepharose. Bound material was eluted with SDS and analysed by immunoblotting with anti-brain spectrin. Lane 1, CC1-P1; Lane 2, CC1-P2; Lane 3, CC1-P3; Lane 4, CC1-P4; Lane 5, no peptide. Note that spectrin bound to Pep1. (e) Competition between a 29mer peptide representing part of the linker region from βIIΣI and brain spectrin. CC1-Sepharose was mixed with brain extract in the absence (Lane 1) or presence (Lane 2) of the 29mer. Bound material was recovered and analysed by immunoblotting with anti-brain spectrin. Note that in the absence of peptide, spectrin was recovered, but that in the presence of peptide no spectrin was recovered. Lane 3 shows a control with CC1-Sepharose processed as in lane 1, but without added brain extract. (f) Binding between the spectrin 29mer and CC1. His-tagged CC1 was immobilised in 96-well plates and incubated with biotinylated 29mer. Binding was detected using streptavidin-peroxidase. The curve is calculated for a single class of binding site, KD 650 nM.
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fig02: Interaction of calmodulin regulated spectrin-associated protein 1 (CAMSAP1) with spectrin. (a) Generalized structures of spectrin and C-terminal variants of β-spectrins. Spectrin α and β subunits are arranged in tetramers. At the C-terminal end of β chains, splice variation gives rise to long (βIIΣI) and short (βIIΣ2) variants. The long variant has a PH domain joined to the last of the helical repeats (repeats 16 and 17) by a linker of around 90 amino acids. Differential mRNA splicing gives rise to a variant in which this linker is interrupted such that the PH domain is lost and the second half of the linker is replaced with a unique (probably unstructured) region in the short variant. (b) Affinity chromatography of CAMSAP1 on various spectrin constructs coupled to Sepharose. A his-tagged central region construct (see Fig 1a) was flowed over the indicated spectrin protein or fragment coupled to Sepharose. Unbound material was washed away, and bound material recovered by elution with 1 M KI. Fractions representing bound material were analysed by immunoblot: sequential fractions from the 1M KI elution, labelled 1–4, are shown here. Note that the CAMSAP1 fragment bound to whole brain spectrin, the βIIΣI C-terminal fragment from repeat 16 to the C-terminus, the βIIΣI linker fragment, but not to the fragment of βIIΣ2 repeat 16 to C-terminus or the PH domain alone. (c) Interaction of brain spectrin with the CAMSAP-conserved region 1 (CC1) region. His-tagged CC1 construct was coupled to Sepaharose and mixed with brain extract. The figure shows an immunoblot of material recovered after washing the beads in buffer and elution with sodium dodecyl sulfate (SDS). Lanes 1-4 show immunoblots probed with antibody to brain spectrin. Lane 1: CC1-Sepharose. Lane 2: bovine serum albumin coupled to Sepharose. Lane 3: Unconjugated Sepharose. Lane 4: AAAA mutant. (d) Interaction of peptides from CC1 with spectrin. Biotinylated peptides CC1-P1-4 (see Methods and Fig 1b) were mixed with brain extract and recovered on streptavidin-Sepharose. Bound material was eluted with SDS and analysed by immunoblotting with anti-brain spectrin. Lane 1, CC1-P1; Lane 2, CC1-P2; Lane 3, CC1-P3; Lane 4, CC1-P4; Lane 5, no peptide. Note that spectrin bound to Pep1. (e) Competition between a 29mer peptide representing part of the linker region from βIIΣI and brain spectrin. CC1-Sepharose was mixed with brain extract in the absence (Lane 1) or presence (Lane 2) of the 29mer. Bound material was recovered and analysed by immunoblotting with anti-brain spectrin. Note that in the absence of peptide, spectrin was recovered, but that in the presence of peptide no spectrin was recovered. Lane 3 shows a control with CC1-Sepharose processed as in lane 1, but without added brain extract. (f) Binding between the spectrin 29mer and CC1. His-tagged CC1 was immobilised in 96-well plates and incubated with biotinylated 29mer. Binding was detected using streptavidin-peroxidase. The curve is calculated for a single class of binding site, KD 650 nM.

Mentions: Figure 2a, summarizes the structure of αII/βII-spectrin tetramers: note that there are two C-terminal splice variants of βII-spectrin. A long C-terminal variant (βIIΣ1) contains a pleckstrin homology domain joined to the last of the helical repeats (the partial repeat 17) via a linker region. A short splice (βIIΣ2) variant arises from differential mRNA splicing that eliminates the PH domain and part of the linker region.


A conserved sequence in calmodulin regulated spectrin-associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth.

King MD, Phillips GW, Bignone PA, Hayes NV, Pinder JC, Baines AJ - J. Neurochem. (2013)

Interaction of calmodulin regulated spectrin-associated protein 1 (CAMSAP1) with spectrin. (a) Generalized structures of spectrin and C-terminal variants of β-spectrins. Spectrin α and β subunits are arranged in tetramers. At the C-terminal end of β chains, splice variation gives rise to long (βIIΣI) and short (βIIΣ2) variants. The long variant has a PH domain joined to the last of the helical repeats (repeats 16 and 17) by a linker of around 90 amino acids. Differential mRNA splicing gives rise to a variant in which this linker is interrupted such that the PH domain is lost and the second half of the linker is replaced with a unique (probably unstructured) region in the short variant. (b) Affinity chromatography of CAMSAP1 on various spectrin constructs coupled to Sepharose. A his-tagged central region construct (see Fig 1a) was flowed over the indicated spectrin protein or fragment coupled to Sepharose. Unbound material was washed away, and bound material recovered by elution with 1 M KI. Fractions representing bound material were analysed by immunoblot: sequential fractions from the 1M KI elution, labelled 1–4, are shown here. Note that the CAMSAP1 fragment bound to whole brain spectrin, the βIIΣI C-terminal fragment from repeat 16 to the C-terminus, the βIIΣI linker fragment, but not to the fragment of βIIΣ2 repeat 16 to C-terminus or the PH domain alone. (c) Interaction of brain spectrin with the CAMSAP-conserved region 1 (CC1) region. His-tagged CC1 construct was coupled to Sepaharose and mixed with brain extract. The figure shows an immunoblot of material recovered after washing the beads in buffer and elution with sodium dodecyl sulfate (SDS). Lanes 1-4 show immunoblots probed with antibody to brain spectrin. Lane 1: CC1-Sepharose. Lane 2: bovine serum albumin coupled to Sepharose. Lane 3: Unconjugated Sepharose. Lane 4: AAAA mutant. (d) Interaction of peptides from CC1 with spectrin. Biotinylated peptides CC1-P1-4 (see Methods and Fig 1b) were mixed with brain extract and recovered on streptavidin-Sepharose. Bound material was eluted with SDS and analysed by immunoblotting with anti-brain spectrin. Lane 1, CC1-P1; Lane 2, CC1-P2; Lane 3, CC1-P3; Lane 4, CC1-P4; Lane 5, no peptide. Note that spectrin bound to Pep1. (e) Competition between a 29mer peptide representing part of the linker region from βIIΣI and brain spectrin. CC1-Sepharose was mixed with brain extract in the absence (Lane 1) or presence (Lane 2) of the 29mer. Bound material was recovered and analysed by immunoblotting with anti-brain spectrin. Note that in the absence of peptide, spectrin was recovered, but that in the presence of peptide no spectrin was recovered. Lane 3 shows a control with CC1-Sepharose processed as in lane 1, but without added brain extract. (f) Binding between the spectrin 29mer and CC1. His-tagged CC1 was immobilised in 96-well plates and incubated with biotinylated 29mer. Binding was detected using streptavidin-peroxidase. The curve is calculated for a single class of binding site, KD 650 nM.
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Related In: Results  -  Collection

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fig02: Interaction of calmodulin regulated spectrin-associated protein 1 (CAMSAP1) with spectrin. (a) Generalized structures of spectrin and C-terminal variants of β-spectrins. Spectrin α and β subunits are arranged in tetramers. At the C-terminal end of β chains, splice variation gives rise to long (βIIΣI) and short (βIIΣ2) variants. The long variant has a PH domain joined to the last of the helical repeats (repeats 16 and 17) by a linker of around 90 amino acids. Differential mRNA splicing gives rise to a variant in which this linker is interrupted such that the PH domain is lost and the second half of the linker is replaced with a unique (probably unstructured) region in the short variant. (b) Affinity chromatography of CAMSAP1 on various spectrin constructs coupled to Sepharose. A his-tagged central region construct (see Fig 1a) was flowed over the indicated spectrin protein or fragment coupled to Sepharose. Unbound material was washed away, and bound material recovered by elution with 1 M KI. Fractions representing bound material were analysed by immunoblot: sequential fractions from the 1M KI elution, labelled 1–4, are shown here. Note that the CAMSAP1 fragment bound to whole brain spectrin, the βIIΣI C-terminal fragment from repeat 16 to the C-terminus, the βIIΣI linker fragment, but not to the fragment of βIIΣ2 repeat 16 to C-terminus or the PH domain alone. (c) Interaction of brain spectrin with the CAMSAP-conserved region 1 (CC1) region. His-tagged CC1 construct was coupled to Sepaharose and mixed with brain extract. The figure shows an immunoblot of material recovered after washing the beads in buffer and elution with sodium dodecyl sulfate (SDS). Lanes 1-4 show immunoblots probed with antibody to brain spectrin. Lane 1: CC1-Sepharose. Lane 2: bovine serum albumin coupled to Sepharose. Lane 3: Unconjugated Sepharose. Lane 4: AAAA mutant. (d) Interaction of peptides from CC1 with spectrin. Biotinylated peptides CC1-P1-4 (see Methods and Fig 1b) were mixed with brain extract and recovered on streptavidin-Sepharose. Bound material was eluted with SDS and analysed by immunoblotting with anti-brain spectrin. Lane 1, CC1-P1; Lane 2, CC1-P2; Lane 3, CC1-P3; Lane 4, CC1-P4; Lane 5, no peptide. Note that spectrin bound to Pep1. (e) Competition between a 29mer peptide representing part of the linker region from βIIΣI and brain spectrin. CC1-Sepharose was mixed with brain extract in the absence (Lane 1) or presence (Lane 2) of the 29mer. Bound material was recovered and analysed by immunoblotting with anti-brain spectrin. Note that in the absence of peptide, spectrin was recovered, but that in the presence of peptide no spectrin was recovered. Lane 3 shows a control with CC1-Sepharose processed as in lane 1, but without added brain extract. (f) Binding between the spectrin 29mer and CC1. His-tagged CC1 was immobilised in 96-well plates and incubated with biotinylated 29mer. Binding was detected using streptavidin-peroxidase. The curve is calculated for a single class of binding site, KD 650 nM.
Mentions: Figure 2a, summarizes the structure of αII/βII-spectrin tetramers: note that there are two C-terminal splice variants of βII-spectrin. A long C-terminal variant (βIIΣ1) contains a pleckstrin homology domain joined to the last of the helical repeats (the partial repeat 17) via a linker region. A short splice (βIIΣ2) variant arises from differential mRNA splicing that eliminates the PH domain and part of the linker region.

Bottom Line: Calmodulin regulated spectrin-associated protein 1 (CAMSAP1) is a vertebrate microtubule-binding protein, and a representative of a family of cytoskeletal proteins that arose with animals.Biol.We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin-binding to neurite outgrowth.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Kent, Kent, UK.

Show MeSH
Related in: MedlinePlus