Serine 16 of stathmin as a cytosolic target for Ca2+/calmodulin-dependent kinase II after CD2 triggering of human T lymphocytes

We investigated specific signaling events initiated after T cell triggering through the costimulatory surface receptors CD2 and CD28 as compared with activation via the Ag receptor (TCR/CD3). We therefore followed the phosphorylation of stathmin, a ubiquitous cytoplasmic phosphoprotein proposed as a general relay integrating diverse intracellular signaling pathways through the combinatorial phosphorylation of serines 16, 25, 38, and 63, the likely physiologic substrates for Ca2+/calmodulin (CaM)-dependent kinases, mitogen-activated protein (MAP) kinase, cyclin-dependent kinases (cdks), and protein kinase A, respectively. We addressed the specific protein kinase systems involved in the CD2 pathway of T cell activation through the analysis of stathmin phosphorylation patterns in exponentially growing Jurkat T cells, as revealed by phosphopeptide mapping. Stimulation via CD2 activated multiple signal transduction pathways, resulting in phosphorylation of distinct sites of stathmin, the combination of which only partially overlaps the CD3- and CD28-induced patterns. The partial redundancy of the three T cell activation pathways was evidenced by the phosphorylation of Ser25 and Ser38, substrates of MAP kinases and of the cdk family kinase(s), respectively. Conversely, the phosphorylation of Ser16 of stathmin was observed in response to both CD2 and CD28 triggering, but not CD3 triggering, with a kinetics compatible with the lasting activation of CaM kinase II in response to CD2 triggering. In vitro, Ser16 of recombinant human stathmin was phosphorylated also by purified CaM kinase II, and in vivo, CaM kinase II activity was indeed stimulated in CD2-triggered Jurkat cells. Altogether, our results favor an association of CaM kinase II activity with costimulatory signals of T lymphocyte activation and phosphorylation of stathmin on Ser16.     

Studies on the site of phosphorylation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase

The phosphorylation site(s) involved in the activation of CaM-kinase IV by CaM-kinase kinase alpha was studied using a mutant CaM-kinase IV (K71R) in which Lys71 (ATP-binding site) was replaced with Arg, because the autophosphorylation of CaM-kinase IV occurring at multiple sites made it difficult to study phosphorylation of the enzyme by CaM-kinase kinase. Sequence analysis of the phosphopeptide from the trypsin digest of CaM-kinase IV (K71R) phosphorylated by CaM-kinase kinase alpha suggested that the phosphorylation of CaM-kinase IV by CaM-kinase kinase only occurred at Thr196. The recombinant mutant CaM-kinase IV in which Thr196 or Thr200 was replaced with nonphosphorylatable alanine showed little activity in the presence and absence of the kinase kinase. The mutant enzyme in which Thr196 was replaced with negatively charged aspartic acid showed almost 25 times as high activity as the wild-type enzyme in the absence of the kinase kinase, and no more activation was observed in its presence. In contrast, the enzyme in which Thr200 was replaced with aspartic acid showed little enzyme activity. Thus, it may be concluded that the phosphorylation of Thr196 in CaM-kinase IV by CaM-kinase kinase is necessary for the subsequent autophosphorylation and activation of CaM-kinase IV.     

Brain-derived neurotrophic factor increases Ca2+/calmodulin-dependent protein kinase 2 activity in hippocampus

Here we show that brain-derived neurotrophic factor (BDNF) stimulates both the phosphorylation of the Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) and its kinase activity in rat hippocampal slices. In addition, we find that: (i) the time course of BDNF action is not accompanied by a change in the spectrum of either alpha- and beta-subunits of CaMK2 detected by immunoblotting; (ii) both treatment of solubilized CaMK2 with alkaline phosphatase and treatment of immunoprecipitated CaMK2 with protein phosphatase 1 reverse phosphorylation and activation of the kinase; (iii) phospholipase C inhibitor D609 and intracellular Ca2+ chelation by 1,2-bis-(o-aminophenoxy)ethane-N,N,N",N',-tetracetic acid tetra(acetoxymethyl)ester or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate but not omission of Ca2+ or Ca2+ chelation by EGTA, abolish the stimulatory effect of BDNF on phosphorylation and activation of CaMK2. These results strongly suggest that the conversion of CaMK2 into its active, autophosphorylated form, but not its concentration, is increased by BDNF via stimulation of phospholipase C and subsequent intracellular Ca2+ mobilization.     

Calcium-stimulated phosphorylation of MAP-2 in pancreatic betaTC3-cells is mediated by Ca2+/calmodulin-dependent kinase II

An understanding of the role of CaM kinase II in the pancreatic beta-cell is dependent on the identification of its cellular targets. One of the best substrates of CaM kinase II in vitro that could function in secretory events is the microtubule-associated protein, MAP-2. By immunoblot analysis, a high molecular weight protein with electrophoretic properties characteristic of MAP-2, was identified in rat insulinoma betaTC3 cells and isolated rat islets. In immunoprecipitation experiments employing alpha-toxin-permeabilized betaTC3 cells, elevation of intracellular Ca2+ or addition of forskolin, an adenylate cyclase activator, induced significant phosphorylation of MAP-2 in situ. The effect of Ca2+ was rapid, concentration-dependent and closely correlated with activation of CaM kinase II under similar experimental conditions. H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (PKA), prevented forskolin-induced MAP-2 phosphorylation but had little effect on MAP-2 phosphorylation stimulated by elevated Ca2+. Phosphopeptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the betaTC3 cells with increased free Ca2+, was strikingly similar to that generated in vitro by CaM kinase II, most notably with regard to the increased phosphate incorporated into one prominent site. These data provide evidence that MAP-2 is phosphorylated by CaM kinase II in the pancreatic beta-cell in situ, and that this event may provide an important link in the mediation of Ca2+-dependent insulin secretion.     

Cortactin-Src kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway.     

Phosphorylation-dependent reversible translocation of Ca2+/calmodulin-dependent protein kinase II to the postsynaptic densities

The translocation of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) to postsynaptic densities (PSDs) was investigated. When soluble CaM kinase II previously autophosphorylated was incubated with PSDs, the kinase was precipitated by centrifugation, indicating that the soluble kinase associated with PSDs and formed a PSD-CaM kinase II complex. Ca2+-independent activity generated by autophosphorylation of the kinase was retained in the complex. A number of PSD proteins were phosphorylated by the kinase associated with PSDs in both the absence and presence of Ca2+. When PSD-CaM kinase II complex was incubated at 30 degrees C, the enzyme was dephosphorylated and released from the complex. These results indicate that CaM kinase II reversibly translocates to PSDs in a phosphorylation-dependent manner.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.     

Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase

Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to beta-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (approximately 1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by approximately 50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport.     

Distribution of Ca2+/calmodulin-dependent protein kinase kinase alpha in the rat central nervous system: an immunohistochemical study

Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated by CaM-kinase IV kinase. We provided a rabbit antiserum against 20 amino acid residues at the carboxyl-terminal end of CaM-kinase IV kinase, and examined regional and intracellular distribution of CaM-kinase IV kinase immunohistochemically in the central nervous system of the rat by light and electron microscopy. The immunoreactivity was found in cellular nuclei of virtually all neurons. However, the immunoreactivity was weak in the nuclei of the granule cells in the cerebellar cortex, although the nuclei of the granule cells were reported to contain high CaM-kinase IV activity. Thus, it was suggested that other types of CaM-kinase IV kinase might exist in the cerebellum, and the present CaM-kinase IV kinase was named as CaM-kinase kinase alpha.     

NKRP1A molecule is involved in transendothelial migration of CD4+ human T lymphocytes

Among human CD4+ T lymphocytes, 5-20% express the C-type lectin molecule NKRP1A. Interestingly, CD4+ NKRP1A+ T lymphocytes express high levels of beta 1 and beta 2 integrins, thus representing a T lymphocyte subset that can possibly adhere and migrate through vascular endothelium. Indeed, resting CD4+ NKRP1A+ lymphocytes, differently from the CD4+ NKRP1A- subset, migrated across endothelial cell monolayers in a Transwell chamber system. This transendothelial migration was strongly reduced after pre-treatment with an anti-NKRP1A monoclonal antibody (mAb). In addition, the NKRP1A negative Jurkatt CD4+ T-cell line that had been stably transfected with NKRP1A cDNA, migrated more rapidly and efficiently than untransfected Jurkatt cells. Finally, mAb-mediated cross-linking of NKRP1A molecule in CD4+ T lymphocytes induced the upregulation of the LFA1 Mg2+ binding site as well as beta 1 and beta 2 integrin chains. Altogether, these findings indicate that NKRP1A molecule is involved in transendothelial migration of resting CD4+ T lymphocytes.     

Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase

Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+.calmodulin (CaM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CaM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017-6023]. We report here similar scattering experiments on the CaM.MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine-triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by approximately 12 A), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM.MLCK.pRLC.AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.     

Phosphorylation at the nuclear localization signal of Ca2+/calmodulin-dependent protein kinase II blocks its nuclear targeting

Translocation of protein kinases with broad substrate specificities between different subcellular compartments by activation of signaling pathways is an established mechanism to direct the activity of these enzymes toward particular substrates. Recently, we identified two isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), which are targeted to the nucleus by an alternatively spliced nuclear localization signal (NLS). Here we report that cotransfection with constitutively active mutants of CaM kinase I or CaM kinase IV specifically blocks nuclear targeting of CaM kinase II as a result of phosphorylation of a Ser immediately adjacent to the NLS of CaM kinase II. Both CaM kinase I and CaM kinase IV are able to phosphorylate this Ser residue in vitro, and mutagenesis studies suggest that this phosphorylation is both necessary and sufficient to block nuclear targeting. Furthermore, we provide experimental evidence that introduction of a negatively charged residue at this phosphorylation site reduces binding of the kinase to an NLS receptor in vitro, thus providing a mechanism that may explain the blockade of nuclear targeting that we have observed in situ.     

Nuclear localization of the delta subunit of Ca2+/calmodulin-dependent protein kinase II in rat cerebellar granule cells

To examine the physiological roles of the delta subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase IIdelta) in brain, we examined the localization of CaM kinase IIdelta in the rat brain. A specific antibody to CaM kinase IIdelta1-delta4 isoforms was prepared by immunizing rabbits with a synthesized peptide corresponding to the unique carboxyl-terminal end of these isoforms. The prepared antibody did not recognize the alpha, beta, and gamma subunits, which were each overexpressed in NG108-15 cells. Immunoblot analysis on various regions and the nuclear fractions from rat brains suggested that some isoforms of CaM kinase IIdelta1-delta4 were abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was analyzed by RT-PCR with a primer pair from variable domain 1 to variable domain 2. We detected the three PCR products delta3.1, delta3.4, and delta3 that contained the nuclear localization signal. These CaM kinase IIdelta3 isoforms were localized in the nuclei in transfected NG108-15 cells. Immunohistochemical study suggested the existence of these isoforms in the nuclei in cerebellar granule cells. These results suggest that CaM kinase IIdelta3 isoforms are involved in nuclear Ca2+ signaling in cerebellar granule cells.     

Changes of the expression of protein substrates of Ca2+/calmodulin-dependent protein kinase II in neonate and adult rats

We studied the expression of whole protein substrates of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the forebrain of neonate and adult rats. Protein substrates were determined by phosphorylation of the soluble and particulate fractions by CaM kinase II with [gamma-33P]ATP. Phosphorylated proteins were analyzed by SDS-PAGE and two-dimensional gel electrophoresis. More than 50 endogenous proteins were found to be phosphorylated by CaM kinase II in both soluble and particulate fractions. The expression of about 15 protein substrates increased in the particulate fraction from neonate to adult rats, and that of several proteins also changed in the soluble fraction. These findings suggest that the expression of protein substrates was regulated during development as well as that of CaM kinase II itself.     

Inhibition of acid secretion in gastric parietal cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93

A novel Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) inhibitor, KN-93 potently inhibits gastric acid secretion from parietal cells. As previously reported (1), treatment of parietal cells with a selective inhibitor of CaM kinase II, KN-62 resulted in the inhibition of cholinergic-stimulated rabbit parietal cell secretion, whereas it failed to inhibit the histamine and forskolin response. In contrast effects of carbachol, histamine and forskolin were significantly inhibited by KN-93 with an IC50 of 0.15, 0.3 and 1 microM, respectively; these effects occurred without any changes in intracellular cyclic AMP and Ca2+ levels. In the present study we investigated the mechanism by which KN-93 acts upon the acid-secreting machinery of gastric parietal cells. Neither redistribution of the proton pump activity nor the morphological transformation were affected by KN-93. The drug only weakly inhibited the H+, K(+)-ATPase activity but strongly dissipated the proton gradient formed in the gastric membrane vesicles and reduced the volume of luminal space. Thus KN-93 acts at pH gradient formation whereas KN-62 acts only at CaM Kinase II.