AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.     

Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase

LIM-kinase 1 (LIMK1) and LIM-kinase 2 (LIMK2) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases. We report our findings that ROCK, a downstream protein kinase of Rho, specifically activates LIMK2 but not LIMK1 downstream of RhoA. LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK (ROCK-Delta3), whereas full-length ROCK selectively activates LIMK2 but not LIMK1. Activation of LIMK2 by RhoA was inhibited by Y-27632, a specific inhibitor of ROCK, but Rac1-mediated activation of LIMK1 was not. ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity. A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes, whereas replacement by glutamate enhanced the protein kinase activity and stress fiber formation by LIMK2. These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization. Together with the finding that LIMK1 is regulated by Pak1, LIMK1 and LIMK2 are regulated by different protein kinases downstream of distinct Rho family GTPases.     

Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein.

Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.     

Changes in intracellular distribution and activity of protein phosphatase PP1gamma2 and its regulating proteins in spermatozoa lacking AKAP4

The second messenger cAMP mediates its intracellular effects in spermatozoa through cAMP-dependent kinase (PKA, formally known as PRKACA). The intracellular organization of PKA in spermatozoa is controlled through its association with A-kinase-anchoring proteins (AKAPs). AKAP4 (A kinase [PRKA] anchor protein 4; also called fibrous sheath component 1 or AKAP 82) is sperm specific and the major fibrous sheath protein of the principal piece of the sperm flagellum. Presumably, AKAP4 recruits PKA to the fibrous sheath and facilitates local phosphorylation to regulate flagellar function. It is also proposed to act as a scaffolding protein for signaling proteins and proteins involved in metabolism. Akap4 gene knockout mice are infertile due to the lack of sperm motility. The fibrous sheath is disrupted in spermatozoa from mutant mice. In this article, we used Akap4 gene knockout mice to study the effect of fibrous sheath disruption on the presence, subcellular distribution, and/or activity changes of PKA catalytic and regulatory subunits, sperm flagellum proteins PP1gamma2 (protein phosphatase 1, catalytic subunit, gamma isoform, formally known as PPP1CC), GSK-3 (glycogen synthase kinase-3), SP17 (sperm autoantigenic protein 17, formally known as SPA17), and other signaling proteins. There were no changes in the presence and subcellular distribution for PP1gamma2, GSK-3, hsp90 (heat shock protein 1, alpha, formally known as HSPCA), sds22 (protein phosphatase 1, regulatory [inhibitor] subunit 7, formally known as PPP1R7), 14-3-3 protein (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein), and PKB (thymoma viral proto-oncogene, also known as AKT) in mutant mice. However, the subcellular distributions for PKA catalytic subunit and regulatory subunits, PI 3-kinase (phosphatidylinositol 3-kinase), and SP17 were disrupted in mutant mice. Furthermore, there was a significant change in the activity and phosphorylation of PP1gamma2 in mutant compared with wild-type spermatozoa. These studies have identified potentially significant new roles for the fibrous sheath in regulating the activity and function of key signaling enzymes.     

Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA

The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoring proteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.     

Dissociation between AKAP3 and PKARII Promotes AKAP3 Degradation in Sperm Capacitation

Ejaculated spermatozoa must undergo a series of biochemical modifications called capacitation, prior to fertilization. Protein-kinase A (PKA) mediates sperm capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine sperm incubated under capacitation conditions. The degradation rate is variable in sperm from different bulls and is correlated with the capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca²⁺-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca²⁺ in the medium or when intracellular Ca²⁺ was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH₄Cl, or when sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKA_RII anchoring during sperm capacitation.     

LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission

Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27^Kip1 expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27^Kip1-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.     

14-3-3 regulates actin dynamics by stabilizing phosphorylated cofilin

The functionality of the actin cytoskeleton depends on a dynamic equilibrium between filamentous and monomeric actin. Proteins of the ADF/cofilin family are essential for the high rates of actin filament turnover observed in motile cells through regulation of actin polymerization/depolymerization cycles. Rho GTPases act through p21-activated kinase-1 (Pak-1) and Rho kinase to inhibit cofilin activity via the LIM kinase (LIMK)-mediated phosphorylation of cofilin on Ser3. We report the identification of 14-3-3zeta as a novel phosphocofilin binding protein involved in the maintenance of the cellular phosphocofilin pool. A Ser3 phosphocofilin binding protein was purified from bovine brain and was identified as 14-3-3zeta by mass spectrometry. The phosphorylation-dependent interaction between cofilin and 14-3-3zeta was confirmed in pulldown and coimmunoprecipitation experiments. Both Ser3 phosphorylation and a 14-3-3 recognition motif in cofilin are necessary for 14-3-3 binding. The expression of 14-3-3zeta increases phosphocofilin levels, and the coexpression of 14-3-3zeta with LIMK further elevates phosphocofilin levels and potentiates LIMK-dependent effects on the actin cytoskeleton. This potentiation of cofilin action appears to be a result of the protection of phosphocofilin from phosphatase-mediated dephosphorylation at Ser3 by bound 14-3-3zeta. Taken together, these results suggest that 14-3-3zeta proteins may play a dynamic role in the regulation of cellular actin structures through the maintenance of phosphocofilin levels.     

Activation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alpha

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through cofilin phosphorylation downstream of distinct Rho family GTPases. Pak1 and ROCK, respectively, activate LIMK1 and LIMK2 downstream of Rac and Rho; however, an effector protein kinase for LIMKs downstream of Cdc42 remains to be defined. We now report evidence that LIMK1 and LIMK2 activities toward cofilin phosphorylation are stimulated in cells by the co-expression of myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), an effector protein kinase of Cdc42. In vitro, MRCKalpha phosphorylated the protein kinase domain of LIM kinases, and the site in LIMK2 phosphorylated by MRCKalpha proved to be threonine 505 within the activation segment. Expression of MRCKalpha induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in cells, whereas MRCKalpha-induced ADF/cofilin phosphorylation was inhibited by the co-expression with the protein kinase-deficient form of LIM kinases. These results indicate that MRCKalpha phosphorylates and activates LIM kinases downstream of Cdc42, which in turn regulates the actin cytoskeletal reorganization through the phosphorylation and inactivation of ADF/cofilin.     

Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop

LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.     

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction

A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.     

Conservation and function of a bovine sperm A-kinase anchor protein homologous to mouse AKAP82.

Protein kinase A regulates sperm motility through the cAMP-dependent phosphorylation of proteins. One mechanism to direct the activity of the kinase is to localize it near its protein substrates through the use of anchoring proteins. A-Kinase anchoring proteins (AKAPs) act by binding the type II regulatory subunit of protein kinase A and tethering it to a cellular organelle or cytoskeletal element. We showed previously that mAKAP82, the major protein of the fibrous sheath of the mouse sperm flagellum, is an AKAP. The available evidence indicates that protein kinase A is compartmentalized to the fibrous sheath by binding mAKAP82. To characterize AKAP82 in bovine sperm, a testicular cDNA library was constructed and used to isolate a clone encoding bAKAP82, the bovine homologue. Sequence analysis showed that the primary structure of bAKAP82 was highly conserved. In particular, the amino acid sequence corresponding to the region of mAKAP82 responsible for binding the regulatory subunit of protein kinase A was identical in the bull. Bovine AKAP82 was present in both epididymal and ejaculated sperm and was localized to the entire principal piece of the flagellum, the region in which the fibrous sheath is located. Finally, bAKAP82 bound the regulatory subunit of protein kinase A. These data support the idea that bAKAP82 functions as an anchoring protein for the subcellular localization of protein kinase A in the flagellum.     

Molecular mechanism of cofilin dephosphorylation by ouabain

We previously reported that phosphorylated cofilin-triosephosphate isomerase (TPI) complex interacts with Na,K-ATPase and enhances the pump activity through the phosphorylation of cofilin via Rho-mediated signaling pathway. In this study, we tested the hypothesis that the dephosphorylation of cofilin may be induced through Na,K-ATPase inhibition by ouabain. The phosphorylation level of cofilin by ouabain which decreases in a time- and dose-dependent manner in various human cell lines, remains unchanged by pretreatment with Src inhibitor, PP2; epidermal growth factor receptor (EGFR) inhibitor, AG1478; Raf-1 kinase (Raf) inhibitor, GW5074; and ERK kinase (MEK) inhibitor, PD98059, and by transfection of Ras dominant negative mutant (RasN17). This suggests that ouabain dephosphorylates cofilin through the Src/EGFR/Ras/Raf/MEK pathway. Ouabain activates Ras/Raf/MEK pathway, but down-regulates Rho kinase (ROCK)/LIM kinase (LIMK)/cofilin pathway, implying that there may be a cross-talk by ouabain between the Ras/Raf/MEK and the ROCK/LIMK/cofilin pathways. Immunofluorescence and flow cytometry suggest that ouabain-induced active form of cofilin may be involved in cytoskeletal reorganization and cell volume regulation. Thus, these findings demonstrate a new molecular mechanism for the dephosphorylation of cofilin through the inhibition of Na,K-ATPase by ouabain.     

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.     

A kinase-anchoring protein 150 and calcineurin are involved in regulation of acid-sensing ion channels ASIC1a and ASIC2a

Acid-sensing ion channel (ASIC) 1a and ASIC2a are acid-sensing ion channels in central and peripheral neurons. ASIC1a has been implicated in long-term potentiation of synaptic transmission and ischemic brain injury, whereas ASIC2a is involved in mechanosensation. Although the biological role and distribution of ASIC1a and ASIC2a subunits in brain have been well characterized, little is known about the intracellular regulation of these ion channels that modulates their function. Using pulldown assays and mass spectrometry, we have identified A kinase-anchoring protein (AKAP)150 and the protein phosphatase calcineurin as binding proteins to ASIC2a. Extended pulldown and co-immunoprecipitation assays showed that these regulatory proteins also interact with ASIC1a. Transfection of rat cortical neurons with constructs encoding green fluorescent protein- or hemagglutinin-tagged channels showed expression of ASIC1a and ASIC2a in punctate and clustering patterns in dendrites that co-localized with AKAP150. Inhibition of protein kinase A binding to AKAPs by Ht-31 peptide reduces ASIC currents in cortical neurons and Chinese hamster ovary cells, suggesting a role of AKAP150 in association with protein kinase A in ASIC function. We also demonstrated a regulatory function of calcineurin in ASIC1a and ASIC2a activity. Cyclosporin A, an inhibitor of calcineurin, increased ASIC currents in Chinese hamster ovary cells and in cortical neurons, suggesting that activity of ASICs is inhibited by calcineurin-dependent dephosphorylation. These data imply that ASIC down-regulation by calcineurin could play an important role under pathological conditions accompanying intracellular Ca(2+) overload and tissue acidosis to circumvent harmful activities mediated by these channels.     

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.     

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.