Phosphorylated LIM Kinases Colocalize with Gamma-Tubulin in Centrosomes During Early Stages of Mitosis

Abstract

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 g-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 g-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that g-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with g-tubulin and play a role in mitotic spindle assembly.

A functional cooperativity between Aurora A kinase and LIM kinase1

“…This study also partly explains our recent observation showing localization of pLIMK1 T508 to the centrosomes during prophase through telophase. 40 In this study, we noted that pLIMK1 T508 colocalizes with Aur-A to the centrosomes during mitosis. At the centrosomes, upon activation through autophosphorylation at T 288 , Aur-A phosphorylates a number of proteins, including LATS2, 60 NDEL 25 for centrosome maturation, kinesin motor protein Eg5, 61 MCAK 62 for spindle bipolarity and ASAP 63 for spindle formation.…”

“…Previously, we showed that phosphoLIMK1 (T 508 )(pLIMK1) colocalized with γ-tubulin to the centrosomes during early prophase till early telophase, while nonphosphorylated LIMK1 did not localize to the centrosomes. 40 To further elucidate the involvement of LIMK1 during early mitotic phases, we examined the correlative expression pattern of LIMK1 and Aur-A, which is involved in recruitment of γ-tubulin to the centrosomes during early prophase, 23 in PC-3 cells. Immunofluorescence analysis (IFA) showed strong colocalization of pLIMK1 with Aur-A at the centrosomes during early prophase (Fig.…”

“…50,51 Overexpression of LIMK1 leads to cytokinesis defects 51 and formation of multipolar spindles, 52 which are noted in a variety of cancers. 52,53 Although LIMK1 needs to be phosphorylated during early prophase 50 for its targeting to the centrosomes, 40 it is not known which kinase(s) phosphorylates LIMK1 during mitosis. Amano et al showed that LIMK1 is not phosphorylated by ROCK or PAK during mitosis.…”

“…40 Furthermore, LIMK1 was shown to be involved in positioning of mitotic spindles during metaphase through modulation of cortical actin through phosphorylation of cofilin. 41 LIMK1 is a LIM domain containing serine/threonine kinase, which modulates actin and microtubule dynamics and participates in a variety of cellular processes (reviewed in refs.…”

Cytoskeletal Molecules Play a Major Role in Cancer Progression

“…Several studies have demonstrated that LIMK regulates cell cycle via regulation of actin cytoskeleton [14,15]. Dynamic alteration of microfilamental structure is very important in cell invasion, movement, adhesion and change in morphology.…”