Limits...
Cap-Gly proteins at microtubule plus ends: is EB1 detyrosination involved?

Bosson A, Soleilhac JM, Valiron O, Job D, Andrieux A, Moutin MJ - PLoS ONE (2012)

Bottom Line: Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY.Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1.They highlight the specificity of carboxypeptidase toward tubulin.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U836, Université Joseph Fourier, Grenoble, France.

ABSTRACT
Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.

Show MeSH
Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.Western-blot analysis of the indicated control proteins (see figure 1) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL1/2, 1∶20,000), and anti-detyrosinated tubulin (L4, 1∶20,000).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3303835&req=5

pone-0033490-g002: Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.Western-blot analysis of the indicated control proteins (see figure 1) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL1/2, 1∶20,000), and anti-detyrosinated tubulin (L4, 1∶20,000).

Mentions: Altogether our results show that the antibodies we have developed against tyrosinated EB1 and putative detyrosinated EB1 are highly specific and are useful in Western-blot experiments. We then used them to examine the possible occurrence of the detyrosinated form of EB1 in mouse cells and tissues. We analyzed potential EB1 detyrosination in wild type fibroblasts and in brain, a tissue in which normally the majority of α tubulin is detyrosinated (see Figure 2C). As EB1 detyrosination could be mediated either by an unknown carboxypeptidase or by the TCP in unusual conditions where its natural substrate (tyrosinated tubulin) is largely decreased (see Figure 2C), we decided to also examine TTL-deficient fibroblast and brain.


Cap-Gly proteins at microtubule plus ends: is EB1 detyrosination involved?

Bosson A, Soleilhac JM, Valiron O, Job D, Andrieux A, Moutin MJ - PLoS ONE (2012)

Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.Western-blot analysis of the indicated control proteins (see figure 1) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL1/2, 1∶20,000), and anti-detyrosinated tubulin (L4, 1∶20,000).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303835&req=5

pone-0033490-g002: Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.Western-blot analysis of the indicated control proteins (see figure 1) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL1/2, 1∶20,000), and anti-detyrosinated tubulin (L4, 1∶20,000).
Mentions: Altogether our results show that the antibodies we have developed against tyrosinated EB1 and putative detyrosinated EB1 are highly specific and are useful in Western-blot experiments. We then used them to examine the possible occurrence of the detyrosinated form of EB1 in mouse cells and tissues. We analyzed potential EB1 detyrosination in wild type fibroblasts and in brain, a tissue in which normally the majority of α tubulin is detyrosinated (see Figure 2C). As EB1 detyrosination could be mediated either by an unknown carboxypeptidase or by the TCP in unusual conditions where its natural substrate (tyrosinated tubulin) is largely decreased (see Figure 2C), we decided to also examine TTL-deficient fibroblast and brain.

Bottom Line: Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY.Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1.They highlight the specificity of carboxypeptidase toward tubulin.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U836, Université Joseph Fourier, Grenoble, France.

ABSTRACT
Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.

Show MeSH