Phosphorylated LIM kinases colocalize with gamma-tubulin in centrosomes during early stages of mitosis.
LIM kinases (LIMK1 and LIMK2) are LIM domain containing serine/threonine kinases that modulate reorganization of actin cytoskeleton through inactivating phosphorylation of cofilin. The Rho family of small GTPases regulates the catalytic activity of LIMK1 and LIMK2 through activating phosphorylation by ROCK or by p21 kinase. Recent studies have suggested that LIMK1 could play a role in modulation of cellular growth by alteration of the cell cycle in breast and prostate tumor cells; however, the direct mitogenic effects of LIMK1 in these tumor cells is yet to be elucidated. Via immunofluorescence, in this study, we show that phosphorylated LIM kinases (pLIMK1/2) are colocalized with gamma-tubulin in the centrosomes during the early mitotic phases of human breast and prostate cancer cells (MDA-MB-231 and DU145); apparent colocalization begins in the centrosomes in prophase. As shown by both bright field (MDA-MB-231) and fluorescent immunohistochemistry (MDA-MB-231 and DU145), pLIMK1/2 does not localize to centrosomes during interphase. By bright field immunohistochemistry, the largest area of the centrosome that is stained with pLIMK1/2 occurs at anaphase. In early telophase, reduced staining of pLIMK1/2 at the spindle poles and concomitant accumulation of pLIMK1/2 at the cleavage furrow begins to occur. In late telophase, loss of staining of pLIMK1/2 and of colocalization with gamma-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that gamma-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with gamma-tubulin and play a role in mitotic spindle assembly.
Chakrabarti
Ratna
R
Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, Florida 32826, USA. rchak@mail.ucf.edu
Jones
Jennifer L
JL
Oelschlager
Denise K
DK
Tapia
Tenekua
T
Tousson
Albert
A
Grizzle
William E
WE
D045744:Cell Line, Tumor; D018385:Centrosome / Q000737:chemistry*; D006801:Humans; D007399:Interphase; D054461:Lim Kinases / Q000378:metabolism*; D008938:Mitosis; D010766:Phosphorylation; D021381:Protein Transport; D008941:Spindle Apparatus; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; C515892:LIMK1 protein, human; C102895:LIMK2 protein, human; D054461:Lim Kinases