Active transport of the Ubiquitin Ligase MID1 along the microtubules Is regulated by Protein Phosphatase 2A

TY - JOUR

T1 - Active transport of the Ubiquitin Ligase MID1 along the microtubules Is regulated by Protein Phosphatase 2A

AU - Aranda-Orgilles, Beatriz

AU - Aigner, Johanna

AU - Kunath, Melanie

AU - Lurz, Rudi

AU - Schneider, Rainer

AU - Schweiger, Susann

PY - 2008/10/24

Y1 - 2008/10/24

N2 - Mutations in the MID1 protein have been found in patients with Opitz BBB/G syndrome (OS), which is characterised by multiple malformations of the ventral midline. MID1 is a microtubule-associated protein that stabilizes microtubules and, in association with the regulatory subunit of protein phosphatase 2A (PP2A), alpha 4, provides ubiquitin ligase activity for the ubiquitin-specific modification of PP2A. Using Fluorescence Recovery After Photobleaching (FRAP) technology, we show here that MID1 is actively and bi-directionally transported along the microtubules, and that this movement is directly linked to its MAP kinase and PP2A-mediated phosphorylation status. Intact transport depends on both kinesins and dyneins and is inhibited upon colcemide treatments. MID1 proteins carrying missense mutations in the alpha 4 binding domain still bind the microtubules but cannot be actively transported. Likewise, knock-down of the alpha 4 protein, inhibition of PP2A activity by okadaic acid and fostriecin or the simulation of permanent phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP, while preserving its microtubule-association. In summary, our data uncover an unexpected and novel function for PP2A, its regulatory subunit alpha 4 and PP2A/alpha 4/mTOR signaling in the active transport of the MID1 ubiquitin ligase complex along the cytoskeleton. Furthermore, a failure in the microtubule directed transport of this protein complex would be an attractive mechanism underlying the pathogenesis of OS in patients with B-box1 mutations.

AB - Mutations in the MID1 protein have been found in patients with Opitz BBB/G syndrome (OS), which is characterised by multiple malformations of the ventral midline. MID1 is a microtubule-associated protein that stabilizes microtubules and, in association with the regulatory subunit of protein phosphatase 2A (PP2A), alpha 4, provides ubiquitin ligase activity for the ubiquitin-specific modification of PP2A. Using Fluorescence Recovery After Photobleaching (FRAP) technology, we show here that MID1 is actively and bi-directionally transported along the microtubules, and that this movement is directly linked to its MAP kinase and PP2A-mediated phosphorylation status. Intact transport depends on both kinesins and dyneins and is inhibited upon colcemide treatments. MID1 proteins carrying missense mutations in the alpha 4 binding domain still bind the microtubules but cannot be actively transported. Likewise, knock-down of the alpha 4 protein, inhibition of PP2A activity by okadaic acid and fostriecin or the simulation of permanent phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP, while preserving its microtubule-association. In summary, our data uncover an unexpected and novel function for PP2A, its regulatory subunit alpha 4 and PP2A/alpha 4/mTOR signaling in the active transport of the MID1 ubiquitin ligase complex along the cytoskeleton. Furthermore, a failure in the microtubule directed transport of this protein complex would be an attractive mechanism underlying the pathogenesis of OS in patients with B-box1 mutations.

KW - LINKED OPITZ-SYNDROME

KW - SYNDROME GENE-PRODUCT

KW - CATALYTIC SUBUNIT

KW - NEURONAL POLARITY

KW - MOTOR PROTEINS

KW - G/BBB SYNDROME

KW - MUTATIONS

KW - BINDING

KW - COMPLEX

KW - DOMAIN

U2 - 10.1371/journal.pone.0003507

DO - 10.1371/journal.pone.0003507

M3 - Article

C2 - 18949047

SN - 1932-6203

VL - 3

SP - -

JO - PLoS ONE

JF - PLoS ONE

IS - 10

M1 - e3507

ER -