LIM Kinase 2 Is Widely Expressed in All Tissues

Abstract

S U M M A R Y The LIM kinase family includes two proteins: LIMK1 and LIMK2. These proteins have identical genomic structure and overall amino acid identity of 50%. Both proteins regulate actin polymerization via phosphorylation and inactivation of the actin depolymerizing factors ADF/cofilin. Although the function of endogenous LIMK1 is well established, little is known about the function of the endogenous LIMK2 protein. To understand the specific role of endogenous LIMK2 protein, we examined its expression in embryonic and adult mice using a rat monoclonal antibody, which recognizes specifically the PDZ domain of LIMK2 but not that of LIMK1. Immunoblotting and immunoprecipitation analyses of mouse tissues and human and mouse cell lines revealed widespread expression of the 75-kDa LIMK2 protein. Immunofluorescence analysis demonstrated that the cellular localization of LIMK2 is different from that of LIMK1. LIMK2 protein is found in the cytoplasm localized to punctae and is not enriched within focal adhesions like LIMK1. Immunohistochemical studies revealed that LIMK2 is widely expressed in embryonic and adult mouse tissues and that its expression pattern is similar to that of LIMK1 except in the testes. We have also demonstrated that endogenous LIMK1 and LIMK2 form heterodimers, and that LIMK2 does not always interact with the same proteins as LIMK1. (J Histochem Cytochem 54:487-501, 2006)

LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs

“…Immunoprecipitation was performed as described earlier (Acevedo et al, 2006). Briefly, whole cell lysates were precleared with protein-G sepharose beads coupled with 2 mg anti-rat IgG2a to remove non-specific binding proteins.…”

“…Equal amounts of protein (15-50 mg) were resolved on 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis or 4-20% pre-cast Criterion Tris-HCl gel (Bio-Rad, Hercules, CA, USA) before electrotransfer. Immunoblotting was performed using anti-LIMK2 mAb (Acevedo et al, 2006), anti-GAPDH (Abcam, Cambridge, UK), anti-b-actin (Sigma-Aldrich), anti-p-LIMK1 (Thr508)/ LIMK2 (Thr505) (Cell Signaling, Beverly, MA, USA), anti-pcofilin (phospho-Ser3), anti-cofilin (Abcam, Cambridge, UK) and anti-g-actin (a gift from P Gunning, University of New South Wales, Australia). The membranes were incubated with HRP-conjugated IgG secondary antibodies and proteins detected with ECL Plus (GE Healthcare Life Sciences, Uppsala, Sweden).…”

“…The distribution pattern of LIMK2 during cell cycle progression is distinct from LIMK1. LIMK2 is expressed as punctae in the cytoplasm of interphase cells (Acevedo et al, 2006), and then accumulates in the centrosomes during prometaphase, then localizes to the mitotic spindle during metaphase and early anaphase and to the spindle midzone at late anaphase and telophase (Sumi et al, 2006). In contrast, LIMK1 is localized in the spindle poles during prometaphase to anaphase and then redistributed to the cleavage furrow in telophase (Sumi et al, 2006).…”

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from theTrans-Golgi Network

“…Practically nothing is known about the regulatory mechanisms involved in the organization of these Golgi filaments and about how these mechanisms contribute to the generation of specific cargo routes exiting the Golgi complex. A clue to actin filament organization at the Golgi may lie in recent studies on LIM kinases (LIMK) 1 and 2, widely expressed down-regulators of the actin-severing activity of cofilin that in neurons are known to operate downstream of cdc42 and PAK1 (Bamburg, 1999; Foletta et al., 2004;Acevedo et al, 2006). Importantly, a study in hypocampal neurons (Rosso et al, 2004) showed that LIMK1 localizes to the Golgi complex via its LIM domains and to axonal growth cones via its postsynaptic density 95/disclarge/zona occludens domains and that overexpression of LIMK1 promotes axonal growth and differentiation.…”

LIMK1/TPPP1/HDAC6 Is a Dual Actin and Microtubule Regulatory Complex That Promotes Drug Resistance

“…An-ti-α-tubulin (T5168), acetyl-α-tubulin (Lys40; T6793) and HDAC6 (SAB1406911) antibodies were purchased from Sigma-Aldrich. Additional antibodies used were TPPP1 [6], LIMK1 [12], LIMK2 [15], Flag 9H1 clone [16] and c-myc tag clone 4A6 (Upstate). Immunoblot quantification was performed using the ImageQuant software.…”