TY - JOUR
T1 - RGS14 is a microtubule-associated protein
AU - Martin-McCaffrey, Luke
AU - Willard, Francis S.
AU - Pajak, Agnieszka
AU - Dagnino, Lina
AU - Siderovski, David P.
AU - D'Souza, Sudhir J.A.
N1 - Funding Information:
(UNC Pharmacology) for assistance with vector During mitosis, microtubules (MTs)—cytoskeletal polymers of α-and β-tubulin het-Harden for the myristoylated Gαi1. We also thankconstruction and protein purification and Dr. Ken erodimers—reorganize into a bipolar spindle that links chromosomes to centrosomes and Drs. Hyeseon Cho and John Kehrl for providing also form into asters that radiate from the spindle poles and attach centrosomes to the cell their anti-RGS14 antisera and mouse-RGS14-GFP. cortex. Tubulins are guanine nucleotide-binding proteins (G-proteins), which exist in either L.M.M. is a recipient of an Ontario Graduate a GDP-or a GTP-bound state. Polymerization/depolymerization dynamics of microtubules Scholarship in Science and Technology. F.S.W. is a are governed by the nucleotide state of β-tubulin.1The presence of a “GTP-cap” promotes postdoctoral fellow of the American Heart microtubule polymerization, whereas depolymerization is induced by hydrolysis of GTP Association. S.J.A.D. is a Canadian Institutes of to GDP or sequestration of free tubulin dimers.2,3 Like many G-proteins, tubulin has Health Research New Investigator. This work was inherent guanosine-triphosphatase (GTPase) activity that is modulated by several different supported by NIH grant R01 GM062338 (to proteins to regulate microtubule dynamics.
PY - 2005/7
Y1 - 2005/7
N2 - Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule-associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin coimmunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 copurifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD = 1.3 ± 0.3 μM). Both RGS14 and Gαi1 in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule-associated proteins. However, preincubation of RGS14 with Gαi1-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule-associated protein that may modulate microtubule dynamics and spindle formation.
AB - Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule-associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin coimmunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 copurifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD = 1.3 ± 0.3 μM). Both RGS14 and Gαi1 in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule-associated proteins. However, preincubation of RGS14 with Gαi1-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule-associated protein that may modulate microtubule dynamics and spindle formation.
KW - Cell division
KW - Heterotrimeric G-protein signaling
KW - Microtubule dynamis
KW - Microtubule-associated protein (MAP)
KW - Mitosis
KW - RGS14
KW - Regulator of G-protein signaling
KW - Tubulin
UR - http://www.scopus.com/inward/record.url?scp=25444483368&partnerID=8YFLogxK
U2 - 10.4161/cc.4.7.1787
DO - 10.4161/cc.4.7.1787
M3 - Article
C2 - 15917656
AN - SCOPUS:25444483368
VL - 4
SP - 953
EP - 960
JO - Cell Cycle
JF - Cell Cycle
SN - 1538-4101
IS - 7
ER -