Cofilin Phosphorylation by Protein Kinase Testicular Protein Kinase 1 and Its Role in Integrin-mediated Actin Reorganization and Focal Adhesion Formation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.

Regulation of the p21-activated kinase (PAK) by a human Gβ-like WD-repeat protein, hPIP1

The family of p21-activated protein kinases (PAKs) is composed of serine–threonine kinases whose activity is regulated by the small guanosine triphosphatases (GTPases) Rac and Cdc42. In mammalian cells, PAKs have been implicated in the regulation of mitogen-activated protein cascades, cellular morphological and cytoskeletal changes, neurite outgrowth, and cell apoptosis. Although the ability of Cdc42 and Rac GTPases to activate PAK is well established, relatively little is known about the negative regulation of PAK or the identity of PAK cellular targets. Here, we describe the identification and characterization of a human PAK-interacting protein, hPIP1. hPIP1 contains G protein β-like WD repeats and shares sequence homology with the essential fission yeast PAK regulator, Skb15, as well as the essential budding yeast protein, MAK11. Interaction of hPIP1 with PAK1 inhibits the Cdc42/Rac-stimulated kinase activity through the N-terminal regulatory domains of PAK1. Cotransfection of hPIP1 in mammalian cells inhibits PAK-mediated c-Jun N-terminal kinase and nuclear factor κ B signaling pathways. Our results demonstrate that hPIP1 is a negative regulator of PAK and PAK signaling pathways.

Identification of GIT1/Cat-1 as a substrate molecule of protein tyrosine phosphatase ζ/β by the yeast substrate-trapping system

We used a genetic method, the yeast substrate-trapping system, to identify substrates for protein tyrosine phosphatases ζ (PTPζ/RPTPβ). This method is based on the yeast two-hybrid system, with two essential modifications: conditional expression of protein tyrosine kinase v-src (active src) to tyrosine-phosphorylate the prey proteins and screening by using a substrate-trap mutant of PTPζ (PTPζ-D1902A) as bait. By using this system, several substrate candidates for PTPζ were isolated. Among them, GIT1/Cat-1 (G protein-coupled receptor kinase-interactor 1/Cool-associated, tyrosine-phosphorylated 1) was examined further. GIT1/Cat-1 bound to PTPζ-D1902A dependent on the substrate tyrosine phosphorylation. Tyrosine-phosphorylated GIT1/Cat-1 was dephosphorylated by PTPζ in vitro. Immunoprecipitation experiments indicated that PTPζ-D1902A and GIT1/Cat-1 form a stable complex also in mammalian cells. Immunohistochemical analyses revealed that PTPζ and GIT1/Cat-1 were colocalized in the processes of pyramidal cells in the hippocampus and neocortex in rat brain. Subcellular colocalization was further verified in the growth cones of mossy fibers from pontine explants and in the ruffling membranes and processes of B103 neuroblastoma cells. Moreover, pleiotrophin, a ligand for PTPζ, increased tyrosine phosphorylation of GIT1/Cat-1 in B103 cells. All these results indicate that GIT1/Cat-1 is a substrate molecule of PTPζ.

Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.

Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs.

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors.

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both RAC-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKalpha activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.

Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Serine-1321-independent regulation of the mu 1 adult skeletal muscle Na+ channel by protein kinase C.

The adult skeletal muscle Na+ channel mu1 possesses a highly conserved segment between subunit domains III and IV containing a consensus protein kinase C (PKC) phosphorylation site that, in the neuronal isoform, acts as a master control for "convergent" regulation by PKC and cAMP-dependent protein kinase. It lacks an approximately 200-aa segment between domains I and II though to modulate channel gating. We here demonstrate that mu1 is regulated by PKC (but not cAMP-dependent protein kinase) in a manner distinct from that observed for the neuronal isoforms, suggesting that under the same conditions muscle excitation could be uncoupled from motor neuron input. Maximal phosphorylation by PKC, in the presence of phosphatase inhibitors, reduced peak Na+ currents by approximately 90% by decreasing the maximal conductance, caused a -15 mV shift in the midpoint of steady-state inactivation, and caused a slight speeding of inactivation. Surprisingly, these effects were not affected by mutation of the conserved serine (serine-1321) in the interdomain III-IV loop. the pattern of current suppression and gating modification by PKC resembles the response of muscle Na+ channels to inhibitory factors present in the serum and cerebrospinal fluid of patients with Guillain-Barré syndrome, multiple sclerosis, and idiopathic demyelinating polyradiculoneuritis.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

The peripheral pro-nociceptive state induced by repetitive inflammatory stimuli involves continuous activation of protein kinase A and protein kinase C epsilon and its Na(v)1.8 sodium channel functional regulation in the primary sensory neuron

In the present study, the participation of the Na(v)1.8 sodium channel was investigated in the development of the peripheral pro-nociceptive state induced by daily intraplantar injections of PGE(2) in rats and its regulation in vivo by protein kinase A (PKA) and protein kinase C epsilon (PKC epsilon) as well. In the prostaglandin E(2) (PGE(2))-induced persistent hypernociception, the Na(v)1.8 mRNA in the dorsal root ganglia (DRG) was up-regulated. The local treatment with dipyrone abolished this persistent hypernociception but did not alter the Na(v)1.8 mRNA level in the DRG. Daily intrathecal administrations of antisense Na(v)1.8 decreased the Na(v)1.8 mRNA in the DRG and reduced ongoing persistent hypernociception. once the persistent hypernociception had been abolished by dipyrone, but not by Na(v)1.8 antisense treatment, a small dose of PGE(2) restored the hypernociceptive plateau. These data show that, after a period of recurring inflammatory stimuli, an intense and prolonged nociceptive response is elicited by a minimum inflammatory stimulus and that this pro-nociceptive state depends on Na(v)1.8 mRNA up-regulation in the DRG. in addition, during the persistent hypernociceptive state, the PKA and PKC epsilon expression and activity in the DRG are up-regulated and the administration of the PKA and PKC epsilon inhibitors reduce the hypernociception as well as the Na(v)1.8 mRNA level. In the present study, we demonstrated that the functional regulation of the Na(v)1.8 mRNA by PKA and PKC epsilon in the primary sensory neuron is important for the development of the peripheral pro-nociceptive state induced by repetitive inflammatory stimuli and for the maintenance of the behavioral persistent hypernociception. (C) 2008 Elsevier Inc. All rights reserved.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

LDLs stimulate p38 MAPKs and wound healing through SR-BI independently of Ras and PI3 kinase.

Intracellular signals elicited by LDLs are likely to play a role in the pathogenesis associated with increased LDL blood levels. We have previously determined that LDL stimulation of human skin fibroblasts, used as a model system for adventitial fibroblasts, activates p38 mitogen-activated protein kinases (MAPKs), followed by IL-8 production and increased wound-healing capacity of the cells. The proximal events triggering these responses had not been characterized, however. Here we show that MAPK kinases MKK3 and MKK6, but not MKK4, are the upstream kinases responsible for the activation of the p38 MAPKs and stimulation of wound closure in response to LDLs. Phosphoinositide 3 kinases (PI3Ks) and Ras have been suggested to participate in lipoprotein-induced MAPK activation. However, specific PI3K inhibitors or expression of a dominant-negative form of Ras failed to blunt LDL-induced p38 MAPK activation. The classical LDL receptor does not participate in LDL signaling, but the contribution of other candidate lipoprotein receptors has not been investigated. Using cells derived from scavenger receptor class B type I (SR-BI) knockout mice or the BLT-1 SR-BI inhibitor, we now show that this receptor is required for LDLs to stimulate p38 MAPKs and to promote wound healing. Identification of MKK3/6 and SR-BI as cellular relays in LDL-mediated p38 activation further defines the signaling events that could participate in LDL-mediated pathophysiological responses.