Inhibition of activated Ras-induced neuronal differentiation of PC12 cells by the LIM domain of LIM-kinase 1

Self Cite

114

103

The regulation of actin cytoskeleton dynamics plays a fundamental role in cell shape change, motility, and migration in response to stimuli. In neurons, actin filaments accumulate at the distal tip of the growth cone in the growing neurite, and actin filament dynamics and reorganization are essential for controlling growth cone motility and morphology and determining the direction and speed of neurite extension (1-4). Cofilin and its closely related protein, actin depolymerizing factor (ADF), 2 are key mediators of actin filament dynamics that act by stimulating the depolymerization and severing of actin filaments (5). The activities of cofilin and ADF are inhibited by phosphorylation at Ser-3 by LIM kinases (LIMKs, composed of LIMK1 and LIMK2) (6, 7) and TES kinases (TESKs, composed of TESK1 and TESK2) (8); the inactive Ser-3-phosphorylated cofilin and ADF (P-cofilin/P-ADF) are reactivated by dephosphorylation by Slingshot (SSH) family protein phosphatases (SSH1, SSH2, and SSH3) (9, 10) and chronophin (a haloacid dehalogenase) (11). Because cofilin/ADF and their upstream regulators, LIMKs and SSHs, are abundant in neuronal growth cones (12-15), these proteins have been implicated in the control of neurite extension and guidance through regulating actin filament dynamics.Neurotrophins are known to regulate neurite outgrowth and guidance (2). Rat pheochromocytoma PC12 cells have been often used as a model system to investigate nerve growth factor (NGF)-induced neurite outgrowth. NGF induces cofilin/ADF dephosphorylation in PC12 cells (16), and overexpression of cofilin/ADF or SSH1 enhances neurite extension from PC12 cells and primary cultured neurons, such as chick dorsal root ganglion (DRG) or rat cortical neurons (13,17). In contrast, overexpression of LIMK1 in neurons suppresses growth cone motility and extension (13). In addition, the growth cone collapse induced by semaphorin-3A (a repulsive guidance molecule) or Nogo-66 (a myelin-associated inhibitor of axon regeneration) requires transient activation of LIMK1 and cofilin phosphorylation in chick DRG neurons (18,19). These results suggest that LIMK1 acts as a negative regulator of neurite outgrowth by inhibiting cofilin/ADF activity. However, recent studies have suggested that LIMK1 has a seemingly opposite function on neurite outgrowth: neurite extension from hippocampal neurons was enhanced by LIMK1 expression and suppressed by blockade of LIMK1 activation (14, 20 -22). Thus, further studies are required to understand the role of LIMK1 in neurite extension.In this study, we examined the roles of cofilin/ADF and its phosphoregulation in neurite extension by knocking down the expression of cofilin/ADF, LIMK1/LIMK2, and SSH1/SSH2 using small interfering RNAs (siRNAs). Knockdown of cofilin/ ADF markedly blocked NGF-induced neurite extension of PC12 cells, and knockdown of SSH1/SSH2 suppressed NGFinduced cofilin/ADF dephosphorylation and neurite extension, indicating that SSH1/SSH2-mediated cofilin/ADF dephosphorylation is crucial for neurite extension. LIMK1...

Section: Discussionsupporting

confidence: 76%

LIM Kinase and Slingshot Are Critical for Neurite Extension

Endo

Ohashi

Mizuno

2007

Self Cite

114

103

“…The non-catalytic domain of LIMK1 has been shown to have effects on cytoskeleton reorganization independent of its kinase activity. Specific regions of the non-catalytic domain (LIM or PDZ) inhibit neurite outgrowth in differentiating PC12 cells in response to Ras, neuronal growth factor (NGF) or a ROCK inhibitor without altering endogenous LIMK1 activity (13,14). These observations suggest that the non-catalytic domain of LIMK1 is involved in regulating cellular processes though protein-protein interaction.…”

mentioning

confidence: 81%

LIM Kinase 1 Is Essential for the Invasive Growth of Prostate Epithelial Cells

Dávila

Frost

Grizzle

et al. 2003

137

Mammalian LIM kinase 1 (LIMK1) is involved in reorganization of actin cytoskeleton through inactivating phosphorylation of the ADF family protein cofilin, which depolymerizes actin filaments. Maintenance of the actin dynamics in an ordered fashion is essential for stabilization of cell shape or promotion of cell motility depending on the cell type. These are the two key phenomena that may become altered during acquisition of the metastatic phenotype by cancer cells. Here we show that LIMK1 is overexpressed in prostate tumors and in prostate cancer cell lines, that the concentration of phosphorylated cofilin is higher in metastatic prostate cancer cells, and that a partial reduction of LIMK1 altered cell proliferation by arresting cells at G 2 /M, changed cell shape, and abolished the invasiveness of metastatic prostate cancer cells. We also show that the ectopic expression of LIMK1 promotes acquisition of invasive phenotype by the benign prostate epithelial cells. Our data provide evidence of a novel role of LIMK1 in regulating cell division and invasive property of prostate cancer cells and indicate that the effect is not mediated by phosphorylation of cofilin. Our study correlates with the recent observations showing a metastasisassociated chromosomal gain on 7q11.2 in prostate cancer, suggesting a possible gain in LIMK1 DNA (7q11.23).LIM kinase 1 (LIMK1) 1 belongs to a novel dual specificity (serine/threonine and tyrosine) kinase family that contain two amino-terminal LIM domains (1). LIMK1 gene is expressed predominantly in brain and in developing neural tissues (2), and its deletion (microdeletion of chromosome 7q11.23) is typical for Williams syndrome (3). Cofilin, one of the actin-binding proteins, considered to be a potent regulator of the actin dynamics (4) by means of its activity in F-actin depolymerization, is the only known substrate of LIMK1. The function of cofilin is inhibited by phosphorylation at the Ser-3 residue (5) by LIMK1, which leads to accumulation of F-actin. The catalytic activity of LIMK1 is regulated by distinct members of the Rho subfamily of small GTPases (Rho, Rac, and Cdc42), which controls actin filament dynamics and focal adhesions assembly in response to extra-and intracellular stimuli. Rho, Rac, and Cdc42 induce formation of stress fibers, assembly of lamellipodia and membrane ruffles, and regulation of filopodial protrusions, respectively (6). LIMK1 has been shown to mediate specifically Rac-induced actin cytoskeleton reorganization and focal adhesion complexes (5, 7, 8). Rac-induced activation of LIMK1 is mediated by PAK1, which phosphorylates LIMK1 on its Thr 508 residue (9). Other studies also proposed that Rhoand Cdc42-induced cytoskeletal changes are mediated through phosphorylation of LIMK1 by Rho-dependent protein kinase ROCK (10) and Cdc42-regulated protein kinase PAK4 (11) and MRCK␣ (12).The non-catalytic domain of LIMK1 contains two tandem repeats of a LIM motif, a putative zinc binding motif, and a PDZ domain, which contains two tandem nuclear exit signal sequence...

“…On the other hand, LIMK1 inhibits neuronal differentiation in rat pheochromocytoma PC12 cells through its LIM domain and by interfering with events downstream of mitogen-activated protein kinase activation (54). In addition, the Rho-associated kinase-dependent pathway is reported to suppress nerve growth factor-induced Rac activation and the neurite outgrowth of PC12 cells (55,56).…”

Section: Discussionmentioning

confidence: 99%

LIM Kinase 1 Activates cAMP-responsive Element-binding Protein during the Neuronal Differentiation of Immortalized Hippocampal Progenitor Cells

Yang

Yoon

Min

et al. 2004

LIM kinase 1 (LIMK1), a novel member of a subclass of the protein-serine/threonine kinases, is known to play a role in the development and maintenance of neuronal circuits that mediate cognitive function. Genetic studies have implicated a mutation of LIMK1 as a causative factor in the impairment of visuospatial cognition in a neurodevelopmental disorder, Williams syndrome. A transcriptional factor, cAMP-responsive element-binding protein (CREB), is thought to be involved in the formation of many types of synaptic plasticity involving learning and memory. In the present study we show that the LIMK1 activity is markedly induced during the differentiation of immortalized hippocampal progenitor (H19-7) cells. We found that the addition of neurogenic growth factor to H19-7 cells induces specific binding between LIMK1 and active CREB, that LIMK1 directly phosphorylates CREB, and that this leads to the stimulation of subsequent cAMP-responsive element-mediated gene transcription during H19-7 cell neuronal differentiation. In addition, we also found that LIMK1 activation occurs through Rac/Cdc42-and p21-activated kinase-mediated signaling pathways. Moreover, when the plasmid encoding kinase-inactive LIMK1 was transfected to block the activation of endogenous LIMK1, the neuronal differentiation of H19-7 cells was significantly suppressed. These findings suggest that LIMK1 activation and subsequent CREB phosphorylation are important in the neuronal differentiation of central nervous system hippocampal progenitor cells.Trophic factors as well as growth factors, which both act by binding to cell surface receptors, are essential for the development of the vertebrate nervous system (1, 2). Trophic factors induce the phosphorylation of their receptor tyrosine kinases, which promote a variety of downstream intracellular signaling pathways and ultimately yield marked changes, and exert long lasting effects, such as those associated with promoting cell growth, survival or death, and proliferation or differentiation. The activation of cellular signaling cascades by growth factors involves the phosphorylation of transcription factors and leads to changes in the expressions of terminal differentiation genes in postmitotic neurons and results in alterations of the neuron mature phenotype. Therefore, transcription factors in neural progenitor cells are important in the establishment of neuronal cell identity.cAMP-responsive element (CRE) 1 -binding protein (CREB) is a central transcription factor that mediates cAMP-responsive genes by binding as a dimer to a conserved CRE motif (3). The CREB plays an important role in cell survival and differentiation by a variety of growth factors (4, 5). Numerous signaling pathways are known to activate CREB, including protein kinase C (6), Ca 2ϩ /calmodulin-dependent protein kinases (7, 8), Ras-dependent p105 kinase (9), p90rsk (10), and Rsk2 (11). In rodents, studies support a role for CREB in learning, memory, and synaptic plasticity (12, 13). The functional specificities of CREB in neuronal deve...