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PubMed2001

Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1.

Lin-11, Isl-1 and Mec-3 (LIM) kinases are serine/threonine kinases that phosphorylate cofilin, an actin depolymerizing protein. LIM kinases have a highly modular structure composed of two N-terminal LIM domains (LIM 1/2), a PSD-95, Dlg and ZO-1 (PDZ) domain and a C-terminal protein kinase domain. Here, we overexpressed individual domains of mouse LIM kinase 1 (LIMK1) in PC12 cells and investigated their effects on neurite outgrowth. Although none of the LIMK1 domains had an effect on spontaneous neurite outgrowth, the N-terminal LIM 1/2 domains strongly inhibited differentiation of PC12 cells after stimulation with both nerve growth factor (NGF) and the Rho-kinase inhibitor Y-27632. In contrast, the overexpressed PDZ domain reduced neurite outgrowth only when differentiation had been induced by Y-27632, but not by NGF. Our data suggest that the different non-catalytic N-terminal domains of LIMK1 contribute to the regulation of neurite extension by using distinct signal transduction pathways.

Amides↗
PubMed1996

Self-association of LIM-kinase 1 mediated by the interaction between an N-terminal LIM domain and a C-terminal kinase domain.

LIM-kinase 1 (LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N-terminus. The LIM motif is thought to be involved in protein-protein interactions. We report here evidence that LIMK1 self-associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMKI revealed that the self-association of LIMK1 was caused by interaction between the N-terminal LIM domain and the C-terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.

Animals↗
PubMed2006

Phosphorylation-dependent regulation of unique nuclear and nucleolar localization signals of LIM kinase 2 in endothelial cells.

LIM kinases (LIMKs) regulate actin dynamics through cofilin phosphorylation and also have a function in the nucleus. Recently we have shown that LIMK2 shuttles between cytoplasm and nucleus in endothelial cells and that nuclear import is inhibited by protein kinase C-mediated phosphorylation of Ser-283. Here we aimed to identify the structural features of LIMK2 responsible for nuclear import. We found that the kinase domain of LIMK2 is localized exclusively in the nucleus and, in contrast to the kinase domain of LIMK1, it accumulated in the nucleolus. Through site-directed mutagenesis, we identified the basic amino acid-rich motif KKRTLRKNDRKKR (amino acids 491-503) as the functional nuclear and nucleolar localization signal of LIMK2. After fusing this motif to enhanced green fluorescent protein, the fusion protein localized exclusively in the nucleus and nucleolus. Mutagenesis studies showed that phosphorylation of Thr-494, a putative protein kinase C phosphorylation site identified within the nuclear localization signal, inhibits nuclear import of the enhanced green fluorescent protein-PDZ kinase domain of LIMK2. After inhibiting nuclear export with leptomycin B, phosphorylation of either Ser-283 or Thr-494 reduced the nuclear import of LIMK2. Phosphorylation of both Ser-283 and Thr-494 sites inhibited nuclear import completely. Our findings identify a unique basic amino acid-rich motif (amino acids 491-503) in LIMK2 which is not present in LIMK1 that serves to target the protein not only to the nucleus but also to the nucleolus. Phosphorylation of Thr-494 within this motif negatively regulates nuclear import of LIMK2.

Active Transport, Cell Nucleus↗
PubMed1997

Mouse LIM-kinase 2 gene: cDNA cloning, genomic organization, and tissue-specific expression of two alternatively initiated transcripts.

LIM-kinase 1 and LIM-kinase 2 (LIMK1 and LIMK2) are members of a novel protein kinase subfamily containing LIM motifs at the N-terminus. There are two isoforms of Limk2 transcripts coding proteins with distinct N-terminal structures: LIMK2a, containing two LIM motifs, and LIMK2b, with one and one-half LIM motifs. Here we report the cDNA and genomic structures of mouse LIMK2. The deduced 638-aminoacid sequence of mouse LIMK2a shows 98% identity with that of rat LIMK2a. The mouse Limk2a gene consists of at least 16 exons and spans more than 50 kb. Exon/intron boundaries of the mouse Limk2a gene are exactly conserved with those of the mouse Limk1 gene. An additional exon encoding the Limk2b-specific 5'-terminal sequence was found to be located between exons 2 and 3, suggesting that Limk2a and 2b mRNAs are transcribed from a single Limk2 gene by an alternative usage of exons near the 5' end of the gene. Limk2a and Limk2b transcripts were expressed at different ratios in a variety of mouse tissues.

Alternative Splicing↗
PubMed2001

Functional involvement of Xenopus LIM kinases in progression of oocyte maturation.

LIM kinases (LIMK), including LIMK1 and LIMK2, are unique LIM-family proteins containing a catalytic (kinase) domain. These kinases phosphorylate an actin-depolymerizing factor, cofilin, involved in the regulation of actin-filament dynamics. An unanswered question is the in vivo function of LIMK and how they contribute to development. When we cloned Xenopus homologues of mammalian LIMK, Xlimk1 and Xlimk2, we found that their mRNA and products were abundantly expressed in oocytes. In addition, we obtained evidence for the functional involvement of Xlimk1/2 during oocyte maturation. The microinjection of Xlimk1/2 mRNA into progesterone-treated oocytes significantly inhibited the appearance of a white maturation spot (WMS), an indicator of entry into meiosis. In oocytes lacking a WMS, the organization and/or migration of the microtubule-derived precursor of the meiotic spindle was predominantly affected. We also found that the ectopic expression of Xlimk1/2 clearly prevented dephosphorylation (activation) of Xenopus cofilin (XAC) during oocyte maturation. Furthermore, co-injection of Xlimk1/2 with the constitutively active type of XAC overcame the inhibitory effects by Xlimk1/2, suggesting that XLIMK-induced abnormality in oocyte maturation was mediated by XAC inactivation. Based on these findings, we propose that XLIMK is a putative regulator of cytoskeletal rearrangements during oocyte maturation, and the interaction between XLIMK activity and microtubule dynamics seems highly likely.

Amino Acid Sequence↗
PubMed1998

Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase.

Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. Here we identify a pathway for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinasel inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augmented cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibited these processes. Our results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis.

3T3 Cells↗
PubMed2006

Lim kinases, regulators of actin dynamics.

The members of the LIM kinase (LIMK) family, which include LIMK 1 and 2, are serine protein kinases involved in the regulation of actin polymerisation and microtubule disassembly. Their activity is regulated by phosphorylation of a threonine residue within the activation loop of the kinase by p21-activated kinases 1 and 4 and by Rho kinase. LIMKs phosphorylate and inactivate the actin depolymerising factors ADF/cofilin resulting in net increase in the cellular filamentous actin. Hsp90 regulates the levels of the LIM kinase proteins by promoting their homo-dimerisation and trans-phosphorylation. Rnf6 is an E3 ubiquitin ligase responsible for LIMK degradation in neurons. The activity of LIMK1 is also required for microtubule disassembly in endothelial cells. While LIMK1 localizes mainly at focal adhesions, LIMK2 is found in cytoplasmic punctae, suggesting that they may have different cellular functions. LIMK1 was shown to be involved in cancer metastasis, while LIMK2 activation promotes cells cycle progression.

Actin Depolymerizing Factors↗
PubMed2006

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration.

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells.

Actins↗
PubMed2006

Rho-kinase regulates tissue morphogenesis via non-muscle myosin and LIM-kinase during Drosophila development.

BACKGROUND: The Rho-kinases (ROCKs) are major effector targets of the activated Rho GTPase that have been implicated in many of the Rho-mediated effects on cell shape and movement via their ability to affect acto-myosin contractility. The role of ROCKs in cell shape change and motility suggests a potentially important role for Rho-ROCK signaling in tissue morphogenesis during development. Indeed, in Drosophila, a single ROCK ortholog, DRok, has been identified and has been found to be required for establishing planar cell polarity. RESULTS: We have examined a potential role for DRok in additional aspects of tissue morphogenesis using an activated form of the protein in transgenic flies. Our findings demonstrate that DRok activity can influence multiple morphogenetic processes, including eye and wing development. Furthermore, genetic studies reveal that Drok interacts with multiple downstream effectors of the Rho GTPase signaling pathway, including non-muscle myosin heavy chain, adducin, and Diaphanous in those developmental processes. Finally, in overexpression studies, we determined that Drok and Drosophila Lim-kinase interact in the developing nervous system. CONCLUSION: These findings indicate widespread diverse roles for DRok in tissue morphogenesis during Drosophila development, in which multiple DRok substrates appear to be required.

Animals↗
PubMed2004

Overexpression of LIM kinase 1 renders resistance to apoptosis in PC12 cells by inhibition of caspase activation.

LIM kinases (LIMKs) regulate actin polymerization by phosphorylating cofilin and are predominantly expressed in neural tissue. In this study, the effect of LIMK1 overexpression in PC12 cell apoptosis was investigated. PC12 cells overexpressing the wild-type LIMK1 were more resistant to serum-withdrawal-induced cell death and the level of caspase 3 activation in these cells was lower than in the control PC12 cells or than in the PC12 cells expressing a mutant LIMK1 lacking the kinase domain. The inhibition of JNK activation was observed in the PC12 cells overexpressing the wild-type LIMK1 after serum withdrawal. These results suggest that the LIMK1 might allow resistance to apoptosis in PC12 cells by inhibiting JNK activation.

Actin Cytoskeleton↗
PubMed2001

Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse.

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

Actin Depolymerizing Factors↗
PubMed2001

LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505.

LIM-kinase 1 and 2 (LIMK1 and LIMK2) phosphorylate cofilin and induce actin cytoskeletal reorganization. LIMK1 is activated by Rho-associated, coiled-coil-forming protein kinase (ROCK) and p21-activated kinase 1 (PAK1), but activation mechanisms and cellular functions of LIMK2 have remained to be determined. We report here that LIMK1 and LIMK2 phosphorylate both cofilin and actin-depolymerizing factor (ADF) specifically at Ser-3 and exhibit partially distinct substrate specificity when tested using site-directed cofilin mutants as substrates. We also show that LIMK2 is activated by ROCK by phosphorylation at Thr-505 within the activation loop. Wild-type LIMK2, but not its mutant (T505V) with replacement of Thr-505 by Val, was activated by ROCK in vitro and in vivo. LIMK2 mutants with replacement of Thr-505 by one or two Glu residues (T505E or T505EE) increased the kinase activity about 3.6-fold but were not further activated by ROCK. When expressed in HeLa cells, wild-type LIMK2, but not the T505V mutant, induced the formation of stress fibres, focal adhesions and membrane blebs. Furthermore, inhibitors of Rho and ROCK significantly suppressed LIMK2-induced stress fibres and membrane blebs. These results suggest that LIMK2 functions downstream of the Rho-ROCK signalling pathway and plays a role in reorganization of actin filaments and membrane structures, by phosphorylating cofilin/ADF proteins.

Actin Depolymerizing Factors↗
PubMed2005

The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones.

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.

Actins↗
PubMed1998

Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.

Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at 3; however, the kinases responsible for this phosphorylation have not been identified. Here we show that LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, our results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin.

Actin Depolymerizing Factors↗
PubMed2006

Hsp90 increases LIM kinase activity by promoting its homo-dimerization.

LIM kinase 1 (LIMK1) is a serine protein kinase that regulates the actin cytoskeleton by phosphorylation and inactivation of actin depolymerizing factor cofilin. LIMK1 activity is regulated by the Rho-GTPases via their serine/threonine kinase effectors Rho-kinase and p21-activated kinases 1 and 4 that phosphorylate LIMK1 on threonine 508 in its activation loop. The purpose of this study was to elucidate the pathway leading to the stability of LIMK1, a protein with a half-life of approximately 20 h. Because the half-life of kinase-dead LIMK1 is only 4 h, it is suggestive that trans- or auto-phosphorylation is responsible for the stabilization of LIMK1. Using known Hsp90 inhibitors, we have shown that the half-life of LIMK1 in cells depends on the presence of active Hsp90. Furthermore, endogenous LIMK1 coimmunoprecipitated with endogenous Hsp90 and this interaction promoted LIMK1 homodimer formation as seen by cross-linking experiments. Hsp90 binds LIMK1 via a recognition sequence within the LIMK1 kinase domain, homologous to that of ErbB-2. Mutation of a proline residue within this sequence to glutamic acid reduces its interaction with Hsp90, inhibits homodimer formation, and reduces its half-life to 4 h. These findings implicate Hsp90 in the stabilization of LIMK1 by promoting homodimer formation and transphosphorylation.

Amino Acid Substitution↗
PubMed2004

Regulation of ADF/cofilin phosphorylation and synaptic function by LIM-kinase.

To investigate the role of the LIM-kinase (LIMK) family in the regulation of ADF/cofilin phosphorylation and synaptic function in the mammalian central nervous system (CNS), we conducted biochemical and electrophysiological analysis using mice that were genetically altered in the expression of LIMK-1 and LIMK-2. We showed here that while LIMK-2 knockout mice exhibited minimal abnormalities, the LIMK-1/2 double knockout mice were more severely impaired in both ADF/cofilin phosphorylation and excitatory synaptic function in the CA1 region of the hippocampus. These results indicate a critical role for the LIMK family in the regulation of ADF/cofilin and synaptic function in the brain.

Actin Depolymerizing Factors↗
PubMed1999

Regulation of actin dynamics: The LIM kinase connection.

A signalling pathway has recently been delineated that connects Rho-family GTPases to the cytoskeleton via LIM kinase and the F-actin depolymerising protein cofilin. The existence of this pathway helps to explain some of the effects of LIM kinase and cofilin in the control of actin dynamics.

Actin Depolymerizing Factors↗
PubMed2000

LIM kinase 1 accumulates in presynaptic terminals during synapse maturation.

LIM kinase 1 (LIMK1) is a cytoplasmic protein kinase that is highly expressed in neurons. In transfected cells, LIMK1 binds to the cytoplasmic tail of neuregulins and regulates the breakdown of actin filaments. To identify potential functions of LIMK1 in vivo, we have determined the subcellular distribution of LIMK1 protein within neurons of the rat by using immunomicroscopy. At neuromuscular synapses in the adult hindlimb, LIMK1 was concentrated in the presynaptic terminal. However, little LIMK1 immunoreactivity was detected at neuromuscular synapses before the 2nd week after birth, and most motoneuron terminals were not strongly LIMK1 immunoreactive until the 3rd week after birth. Thus, LIMK1 accumulation at neuromuscular synapses coincided with their maturation. In contrast, SV2, like many other presynaptic terminal proteins, can be readily detected at neuromuscular synapses in the embryo. Similar to its late accumulation at developing synapses, LIMK1 accumulation at regenerating neuromuscular synapses occurred long after these synapses first formed. In the adult ventral spinal cord, LIMK1 was concentrated in a subset of presynaptic terminals. LIMK1 gradually accumulated at spinal cord synapses postnatally, reaching adult levels only after P14. This study is the first to implicate LIMK1 in the function of presynaptic terminals. The concentration of LIMK1 in adult, but not nascent, presynaptic terminals suggests a role for this kinase in regulating the structural or functional characteristics of mature synapses.

Animals↗