The LIMK Isoforms
Several isoforms resulting from alternative splicing were identified for LIMK1 and LIMK2. There are three LIMK1 isoforms: full length LIMK1, LIMK1 short (LIMK1-s) lacking 20 amino acids in the kinase domain rendering it inactive because of modifications to the ATP binding site (Stanyon and Bernard 1999), and a truncated protein dLIMK1, which is devoid of the kinase domain (Edwards and Gill 1999). LIMK2 has two major isoforms that are transcribed from two independent sites, resulting in the full-length LIMK2a and the N-terminal truncated LIMK2b that lacks the first zinc finger of the LIM domain but contains 22 new amino acids acquired from the adjacent 5′ intron (Ikebe et al. 1997). Functional differences between the two LIMK2 isoforms have recently been determined and will be discussed below. Another less studied LIMK2 isoform is tLIMK2, a testis-specific isoform that lacks LIM domains and part of the PDZ domain, suggesting a role of this protein in spermatogenesis (Takahashi et al. 2002).
LIMK Substrates: Function and Regulation
LIMK substrates and general function: The most extensively characterized substrates of LIMK are cofilin 1 (non-muscle cofilin), cofilin 2 (muscle cofilin), and destrin (actin depolymerizing factor or ADF). The ADF/cofilin family of actin depolymerizing factors are central players in the regulation of actin-driven cellular processes such as cytokinesis, membrane ruffling, phagocytosis, cell motility, fluid phase endocytosis, and neurite outgrowth (Bamburg 1999). These small molecular weight (15–20 kDa) proteins (referred to herein as cofilin) are essential for the high turnover rates of actin filaments seen in cells. Cofilin binds to both filamentous actin (F-actin) and globular actin (G-actin). Upon binding to F-actin, it induces a conformational change that leads to a twist in the filament and increases the off rate of actin subunits at the pointed end. These conformational changes increase the thermodynamic instability of the actin polymer which is also responsible for cofilin’s severing activity (Bamburg 1999). Cofilin is also able to form a complex with G-actin; however, it has a greater binding affinity to actin-ADP than actin-ATP. Binding of cofilin to actin-ADP inhibits nucleotide exchange and thus acts as a sequestering protein by reducing the pool of actin-ATP available for polymerization. The combined functions of cofilin (depolymerizing, severing, and sequestering) increase actin turnover by increasing the number of free-barbed ends as well as actin monomers necessary for the assembly of a new filament (Bamburg 1999). Cofilin’s activity is primarily regulated by phosphorylation at the highly conserved serine 3 residue. Phosphorylation at this site inhibits cofilin’s ability to bind to F-actin and to induce its depolymerization, resulting in the accumulation of actin filaments (Moriyama et al. 1996; Nebl et al. 1996). The important discovery that LIMK phosphorylates cofilin and inhibits its activity, resulting in the accumulation of F-actin, identified the members of the LIMK family as important regulators of actin dynamics (Arber et al. 1998; Yang et al. 1998).
Other less studied LIMK substrates are the transcription factors CREB and Nurr1, suggesting a link through LIMK to transcriptional regulation. LIMK activation by FGF leads to increased CREB phosphorylation and CREB-responsive promoter activity (Yang et al. 2004). Nurr1 is an orphan receptor belonging to the nuclear receptor family that regulates gene transcription via hormone response elements in promoter sequences. LIMK1 phosphorylated Nurr1, leading to diminished transcriptional activity from a promoter/reporter construct (Sacchetti et al. 2006).
Another LIMK1 substrate is p25, also known as tubulin polymerization promoting protein (TPPP). When phosphorylated by LIMK1, p25/TPPP loses its ability to promote microtubule (MT) assembly resulting in MT disassembly in vitro and in cells (Acevedo et al. 2007).
LIMK regulation: In the initial studies leading to the discovery that LIMK1 regulates actin dynamics via phosphorylation of cofilin, it was demonstrated that LIMK1 was activated through the Rho-GTPases (Arber et al. 1998; Yang et al. 1998). This identified a novel pathway by which the Rho-GTPases regulate the actin cytoskeletal changes in response to the cellular environment via LIMK1. Furthermore, it was shown that the activity of the LIMK proteins is regulated by the phosphorylation of a threonine residue in the activation loop by the kinases activated by the Rho-GTPases. Both LIMK1 and LIMK2 are phosphorylated by the Rho effectors Rho-associated kinase 1 and 2 (ROCK1 and ROCK2) on the conserved threonine residues, Thr-508 (LIMK1) (Ohashi et al. 2000) and Thr-505 (LIMK2) (Amano et al. 2001; Sumi et al. 2001a). p21-activated kinase (PAK) 1, 2, and 4 were also shown to activate LIMK1 (Edwards et al. 1999; Misra et al. 2005; Dan et al. 2001). PAK 4 activates LIMK1 not only through phosphorylation of Thr-508 but also through phosphorylation and inactivation of the LIMK phosphatase slingshot (see below) (Soosairajah et al. 2005). The myotonic dystrophy kinase-related Cdc42-binding kinase (MRCKα) has been reported to phosphorylate and activate both LIMK1 and LIMK2 (Sumi et al. 2001b). The most studied LIMK activators are ROCK1 and 2. Only limited information on the role of the other kinases mentioned above in LIMK activation is available.
LIMK activity is also regulated by auto- and trans-phosphorylation. Unphosphorylated LIMK1 has a short half-life of ∼4 h, in comparison the phosphorylated protein has a half-life of ∼24 h. LIMK transphosphorylation is mediated by its association with Hsp90 which promotes homodimerization and transphosphorylation, leading to the formation of stable LIMK dimers with increased specific activity (Li et al. 2006).
MAPK/MK2 phosphorylates both Hsp27 and Ser323 of LIMK1, via VEGF-A regulation.
Semaphorin upregulates LIMK2 activity in the presence of plexin C1, a receptor for semaphorin 7A.
Neuregulin interacts with and activates LIMK1.
The interaction between 14-3-3 and LIMK1 increases cofilin phosphorylation by an unknown mechanism.
PKA activates LIMK1 by phosphorylation at serine 323 and 596.
PKC activates LIMK1 by direct interaction.
SDF-1a activates LIMK1.
Nogo-66 activates LIMK1 by a ROCK-dependent phosphorylation.
Ionomycin/CaMKIV activate LIMK1 by direct phosphorylation at Thr508.
The ubiquitin ligases Rnf6 and parkin induce the proteasomal degradation of LIMK.
Nischarin inhibits the Rac/PAK activation of both LIMK1 and LIMK2 by direct association with LIMK.
LATS1 interacts with and reduces LIMK1 activity.
Par-3 interacts with and inhibits LIMK2 activity in vitro and its overexpression suppresses cofilin phosphorylation that is induced by lysophosphatidic acid.
The cytoplasmic tail of BMPR-II interacts with and inhibits the activity of LIMK.
In addition, miR-134 inhibits LIMK1 mRNA translation in neurons.
Specific regulation of the LIMK2 isoforms: Recently, it was reported that p53 induces the expression of LIMK2b, but not that of LIMK2a, after DNA damage by binding to a p53-binding consensus motif in intron 1 of the LIMK2b gene (intron 2 of LIMK2a) (Hsu et al. 2010). The transcription factor p53 is an important cell cycle regulator. After DNA damage, p53 activation induces cell cycle arrest and the activation of pro-survival pathways allowing DNA damage repair. However, with excessive DNA damage, p53 activation may lead to apoptotic cell death. Ectopic p53 expression increases LIMK2b expression and cofilin phosphorylation (Hsu et al. 2010), suggesting that the LIMK2b-mediated actin regulatory function is required for cell cycle arrest. While these results demonstrate that LIMK2b has a role in G2/M cell cycle arrest, the reason for specific p53-mediated LIMK2b induction remains elusive. It is possible that LIMK2b has a distinct role in the cell.
Apart from specific LIMK2b induction following increased p53 levels after DNA damage caused by radiation or drugs such as doxorubicin, no other functional difference between the LIMK2 isoforms have been established to date. Overexpression of LIMK2a or LIMK2b reduced both cell proliferation and viability, and resulted in multi-nucleated cells. These cellular changes were more pronounced with enforced LIMK2b expression, suggesting that the additional 22 amino acids replacing the first zinc-finger may increase LIMK2 activity. Finally, analysis of a large cohort of cell lines by RT-PCR demonstrated that LIMK2a and LIMK2b were differentially expressed in a number of cell lines. Interestingly, the levels of LIMK2a and LIMK2b are downregulated in thyroid cancers while in esophageal cancers LIMK2b is downregulated but LIMK2a levels are increased in comparison to normal tissues (Hsu et al. 2010).
Expression and Cellular Localization of LIMK
LIMK1: Early studies on the expression of LIMK1 mRNA identified it mainly as a brain specific protein (Bernard et al. 1994; Mizuno et al. 1994). However, with the development of good anti-LIMK1 antibodies it was demonstrated that LIMK1 is expressed in all the cells of mouse tissues but at different levels (Foletta et al. 2004). LIMK1 gained special attention due to its high level of expression in cell lines generated from metastatic human breast and prostate tumors, with the notion that LIMK1 may be involved in cancer metastasis (see below).
LIMK2: As for LIMK1, early data on LIMK2 expression in mouse tissues relied on mRNA expression due to the lack of suitable anti-LIMK2 Abs. LIMK2, like LIMK1, is expressed in all mouse tissues (Acevedo et al. 2006). Although both proteins are expressed in both the nuclei and the cytoplasm the subcellular localization of LIMK2 is distinct from that of LIMK1. LIMK2 clearly localizes to punctae resembling endosomes in several cell lines and mouse embryonic fibroblasts (Fig. 3) (Acevedo et al. 2006). In NIH3T3 cells LIMK2 protein is also associated with GM130, a protein associated with the cis-compartment of the Golgi apparatus (Acevedo et al. 2006). Immunofluorescence staining showed that during mitosis the localization of LIMK2 is similar to that of LIMK1 (Gamell-Fulla and Bernard). LIMK2 is associated with the mitotic spindle-like structure during mitosis and accumulates at the centrosomes in prometaphase. During the metaphase to anaphase transition, LIMK2 redistributes to the mitotic spindle and finally to the spindle midzone in telophase (Sumi et al. 2006). The distinct subcellular localization of LIMK2 suggests that it may have different substrates and cellular functions to that of LIMK1.
LIMK1 and Cancer Metastasis
Consistent with the role of LIMK1 in the regulation of the actin cytoskeleton there is growing evidence to indicate that LIMK1 plays an important role in tumor cell invasion and metastasis. High LIMK1 expression was found in metastatic melanoma cells (Wang et al. 2004), breast and prostate tumors (Wang et al. 2004; Davila et al. 2003), and tumor cell lines (Davila et al. 2003; Yoshioka et al. 2003). Several studies have implicated the involvement of LIMK1 in cell invasion and cancer metastasis. Downregulation of LIMK1 activity by expression of dominant-negative LIMK1 in the invasive human breast cancer cell line MDA-MB-231 reduced their invasion in vitro and in mice while overexpression of LIMK1 in the non-invasive MCF7 human breast cancer cells increased their invasion (Yoshioka et al. 2003). Similarly, expression of LIMK1 in MDA-MB-435 human breast cancer cells enhanced their proliferation, invasiveness, and promoted angiogenesis in vitro. When injected to immunocompromised nude mice these cells grew faster, promoted tumor angiogenesis, and induced liver and lung metastasis. (Bagheri-Yarmand et al. 2006). Benign prostate epithelial cells overexpressing LIMK1 exhibit increased expression of Membrane type matrix metalloproteinase 1 (MT1-MMP), a critical modulator of extracellular matrix (ECM) turnover through pericellular proteolysis that plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and LIMK1 are both highly expressed in prostate tumor tissues (Tapia et al. 2011).
A recent study demonstrated that not only LIMK1 but also LIMK2 promote the metastasis of pancreatic cancer cells using a cell-based in vitro migration assay, as well as two zebra fish xenograft assays. The double knock down of LIMK1 and LIMK2 completely blocked invasion and formation of micrometastasis in vivo suggesting that both LIMK proteins have an important role in tumor progression and metastasis formation (Vlecken and Bagowski 2009). Scott et al. demonstrated that inhibition of LIMK activity by the pharmacological agent BMS3 (LIMKi) or by siRNA mediated knockdown blocks the collective invasion of MDA-MB-231 breast carcinoma cells growing in three-dimensional (3D) matrices (Scott et al. 2010). LIMK activity was also required for the collective invasion of squamous carcinoma cells (SCCs) in a 3D organotypic skin model, but it was not required for cell motility in two dimensions (2D). However, as described previously (Bagheri-Yarmand et al. 2006), LIMK was responsible for extracellular matrix (ECM) degradation in 3D cultures but not for path finding in MDA-MB-231 or SCCs (Scott et al. 2010).
The members of the LIM kinase family, LIMK1 and LIMK2 are ubiquitously expressed serine kinases that share identical genomic structure and ∼50% overall identity. The most studied substrates of LIMK1 and LIMK2 are the actin depolymerizing factor ADF/cofilin family of proteins. These actin binding proteins bind to and severe actin filaments (F-actin) and sequester actin monomers resulting in actin depolymerization. Phosphorylation of cofilin by LIMK inhibits its actin binding activity resulting in the accumulation of F-actin. The activity of the LIMK proteins is regulated by the Rho-GTPases: Rho, Rac, and Cdc42 via their downstream effector kinases ROCK 1 and 2, PAK 1, 2, and 4, which phosphorylate LIMK on a threonine residue. Although the LIMK proteins show significant structural similarity, their expression pattern, subcellular localization, regulation, and functions are different. They are involved in many cellular functions, such as cell migration, cell cycle, and neuronal differentiation and also have a role in cancer cell invasion and metastasis. The actin cytoskeleton plays a pivotal role in the motility of normal cells and in the invasive capacity of tumor cells. Both polymerization and depolymerization of actin are required for cell motility and invasion. LIMK1 levels are high in metastatic breast, prostate, and melanoma tumors and in a variety of invasive cell lines. Overexpression of LIMK1 in breast and prostate cancers increased their invasion in vitro and in mice while downregulation of its activity reduced their invasiveness, suggesting that inhibition of LIMK activity with pharmacological agents may be used to inhibit the metastatic spread of cancer cells.
- Scott RW, Hooper S, Crighton D, Li A, Konig I, Munro J, Trivier E, Wickman G, Morin P, Croft DR, Dawson J, Machesky L, Anderson KI, Sahai EA, Olson MF. LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells. J Cell Biol. 2010;191:169–85.PubMedPubMedCentralCrossRefGoogle Scholar