A role for regulated binding of p150Glued to microtubule plus ends in organelle transport

Abstract

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150Glued by live-cell imaging. Time-lapse analysis of p150Glued revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein–glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150Glued phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150Glued phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150Glued phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150Glued during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150Glued–labeled microtubules just prior to transport, implicating microtubules and dynactin in a search–capture mechanism for minus-end–directed organelles.

Arl3 and LC8 regulate dissociation of dynactin from dynein

“…1c,d). Functional importance of this domain to binding with dynein DIC is controversial [11][12][13] . The mechanisms by which Arl3 binding to p150 Glued facilitates the disassembly of dynein-dynactin complex is unknown.…”

“…The second coiled-coil domain of p150 Glued aa 925-1050 on coiled coil 2 (CC2) contains a conserved actin-binding motif that has been proposed to bind the Arp1 filament directly 10 . Moreover, p150 Glued aa 217-548 on CC1 interacts with amino-terminal fragments of the dynein IC (DIC) 11 and the aa 600-811 was also reported to bind full-length DIC 12 , but the latter binding is controversial 13 . Thus, p150 Glued functions as a hub for connection of dynein with cargo.…”

Polo-like kinase 1 phosphorylation of p150 Glued facilitates nuclear envelope breakdown during prophase

“…Two studies showed that phosphorylations in CAP-Gly domain of p150 Glued would inhibit its interaction with microtubules and Aurora A, which is a mitotic kinase, was demonstrated to contribute to some of the phosphorylations (13,14). In this communication, we demonstrated that Plk1 is a kinase responsible for phosphorylation of p150 Glued at Ser-179 and Plk1-mediated phosphorylation of p150 Glued at Ser-179 also improves its dissociation from microtubules, and therefore indirectly promotes its NE accumulation at prophase.…”

“…Two studies showed that phosphorylation in CAP-Gly domain of p150 Glued would inhibit its interaction with microtubules (13,14). Considering that S179 also locates very close to the CAP-Gly domain, we therefore examined the affects of Plk1 phosphorylation of p150 Glued on microtubule association.…”

Molecular and Functional Basis for the Scaffolding Role of the p50/Dynamitin Subunit of the Microtubule-associated Dynactin Complex

“…In addition, dynactin plays other roles independently of microtubule motors, by facilitating attachment of microtubules to several subcellular structures. Although initial studies demonstrated that dynactin contributed to microtubule anchoring at the centrosome during interphase (15,16), recent results provided the first clues concerning its role as an initial receptor for cytoplasmic membrane vesicles at microtubule plus ends (17)(18)(19)(20), although this model has been brought into question (21).…”