CPG16, a Novel Protein Serine/Threonine Kinase Downstream of cAMP-dependent Protein Kinase

Abstract

Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested,e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase. Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested,e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase. Learning and memory processes in the brain are characterized by plasticity changes in central nervous system neurons. In a comprehensive search for candidate plasticity-related genes (CPGs 1The abbreviations used are: CPG, candidate plasticity-related gene; 8-Br-cAMP, 8-bromo-cAMP; PKA, cAMP-dependent protein kinase; MAPK, mitogen-activated protein kinase; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CREB, cAMP response element-binding protein; ERK, external signal-responsive kinase; GST, glutathione S-transferase; HA, hemagglutinin; PBS, phosphate-buffered saline; WIS, Weizmann Institute of Science, Israel; FCS, fetal calf serum; MBP, myelin basic protein. (1Nedivi E. Hevroni D. Naot D. Israeli D. Citri Y. Nature. 1993; 363: 718-722Crossref PubMed Scopus (437) Google Scholar, 2Hevroni D. Rattner A. Bundman M. Lederfein D. Gbarah A. Mangelus M. Silverman M.A. Kedar H. Naor C. Kornuc M. Hanoch T. Seger R. Theill L.E. Nedivi E. Richter-Levin G. Citri Y. J. Mol. Neurosci. 1998; 10: 75-98Crossref PubMed Scopus (138) Google Scholar)), a novel cDNA encoding for a putative protein kinase, termedcpg16, has been isolated from kainate-treated rat hippocampus. Here we show that CPG16 is a protein serine/threonine kinase that, when expressed in COS7 cells, is activated by cAMP via a cAMP-dependent protein kinase (PKA)-induced mechanism. Studies on the effect of CPG16 on transcription have revealed that it may be involved in the down-regulation of cAMP response element-binding protein (CREB) activity. Thus, it is possible that CPG16 participates in the regulation of neuronal plasticity by down-regulating PKA-stimulated transcription. Northern blotting was performed as described previously (3Sadot E. Jaaro H. Seger R. Ginzburg I. J. Neurochem. 1998; 70: 428-431Crossref PubMed Scopus (29) Google Scholar) with 5 μg of RNA/lane. The probe used for detection was the 1600-base pair cpg16 cDNA (2Hevroni D. Rattner A. Bundman M. Lederfein D. Gbarah A. Mangelus M. Silverman M.A. Kedar H. Naor C. Kornuc M. Hanoch T. Seger R. Theill L.E. Nedivi E. Richter-Levin G. Citri Y. J. Mol. Neurosci. 1998; 10: 75-98Crossref PubMed Scopus (138) Google Scholar), which was labeled by the random priming technique (U. S. Biochemical Corp.) using [α-32P]dCTP according to the manufacturer's instructions. The polymerase chain reaction method was used to clone the cDNA encoding the open reading frame for cpg16 (2Hevroni D. Rattner A. Bundman M. Lederfein D. Gbarah A. Mangelus M. Silverman M.A. Kedar H. Naor C. Kornuc M. Hanoch T. Seger R. Theill L.E. Nedivi E. Richter-Levin G. Citri Y. J. Mol. Neurosci. 1998; 10: 75-98Crossref PubMed Scopus (138) Google Scholar) in-frame with glutathioneS-transferase (GST) into the pGEX2T expression vector and purified as described (4Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). To construct a hemagglutinin (HA)-cpg16 plasmid, polymerase chain reaction was used to clone the cpg16 in-frame with the HA epitope tag in a pCGN vector (5Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar). The HA-CPG16 open reading frame was sequenced to ensure its composition. COS-7 or NIH-3T3 cells were grown in 6-cm tissue culture plates containing Dulbecco's modified Eagle's medium and 10% fetal calf serum (FCS) to approximately 60% confluency. The cells were then transfected with plasmid DNA, prepared using Qiagen columns (Qiagen). Subsequently, the COS7 cells were transfected by the DEAE-dextran-mediated method as described previously (6Keown W.A. Campbell C.R. Kucherlapati R.S. Methods Enzymol. 1990; 185: 527-537Crossref PubMed Scopus (95) Google Scholar) using 2.5 μg/ml DNA. After 10% Me2SO shock, cells were cultured in normal growth media and maintained in a humidified incubator (5% CO2) at 37 °C. NIH-3T3 cells were transfected by the calcium phosphate method using 5 μg of DNA (7Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). After the transfection, the cells were cultured in normal growth media and maintained in a humidified incubator (5% CO2) at 37 °C. Control and HA-cpg16-transfected COS-7 cells in 6-cm tissue culture plates were starved for 16 h in 0.1% FCS in Dulbecco's modified Eagle's medium. The cells were then treated with either 10 μm forskolin (Sigma), 10 μm8-bromo-cAMP (8-Br-cAMP, Sigma), 10% FCS, or other stimuli for various times. If isobutylmethylxanthine (Sigma, 10 μm) was included in an experiment, it was added 5 min before other treatments. After stimulation, the cells were washed once with ice-cold phosphate-buffered saline (PBS) and lysed (10 min on ice) in Buffer H (50 mm β-glycerophosphate, pH 7.3, 1.5 mmEGTA, 1 mm EDTA, 1 mm dithiothreitol, 0.1 mm sodium vanadate, 1 mm benzamidine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 μg/ml pepstatin-A (8Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar)). The cells were then scraped from the plate and centrifuged (14,000 × g, 20 min, 4 °C). The supernatant containing HA-CPG16 was collected and incubated with 2 μg of anti-HA antibody (4 °C) for 1 h, then precipitated with protein A-agarose (Sigma) for 1 h. The complexes were washed (4 °C) three times with PBS + 0.1% Nonidet P-40, once with 0.5 mLiCl in PBS, and twice with buffer A ((50 mmβ-glycerophosphate, pH 7.3, 1.5 mm EGTA, 1 mmEDTA, 1 mm dithiothreitol, and 0.1 mmNa3VO4 (8Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar)). GST-CPG16 or HA-CPG16 autophosphorylation was performed at 30 °C in a buffer containing 50 mm HEPES, pH 7.5, 10 mm magnesium acetate, 20 μm ATP (20 cpm/fmol), 10 μg/ml leupeptin, 10 μg/ml aprotinin, 50 mm β-glycerophosphate, 100 μm orthovanadate in a final volume of 30 μl. The reactions were terminated by adding SDS-polyacrylamide gel electrophoresis sample buffer and boiled for 5 min. In vitrokinase assays were performed using the same reaction mixture with the following substrates or protein kinases. Recombinant Jun N-terminal kinase, recombinant external-signal responsive kinase (ERK), recombinant p38 mitogen-activated protein kinase (p38MAPK), recombinant MAPK/ERK kinase, and recombinant ΔN-EE-MAPK/ERK kinase were prepared as described (7Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). Activated ERK was prepared by phosphorylation with active MAPK/ERK kinase (7Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). Nuclear K-7 was the kind gift from Dr. K. Bomsztyk, University of Washington, Seattle. Purified vitronectin and PKA (catalytic subunit) were gifts from Dr. S. Shaltiel, The Weizmann Institute of Science, Israel (WIS). GST-Jun 1–74 and GST-ATF2 16–96 as well as GST-Rho and GST-ARF were expressed in bacteria and purified as described (10Levi N.L. Hanoch T. Benard O. Rozenblat M. Harris D. Reiss N. Naor Z. Seger R. Mol. Endocrinol. 1998; 12: 815-824Crossref PubMed Google Scholar). MAP2 was a gift from Dr. Z. Eleazar (WIS). Peptides based on the following sequences were synthesized by the biological services at the WIS: epidermal growth factor receptor 661–675, glycogen synthase kinase 6–20, Jun 60–78, and Far1 134–150. Myosin light chain, histone IIIs, myelin basic protein (MBP), β-casein, and protamine were purchased from Sigma. 32P-Labeled proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (Immobilon-P; Millipore Corp.). Membrane slices containing the proteins were hydrolyzed in constant boiling 5.7 n HCl and analyzed by two-dimensional electrophoresis as described (11Kamps M.P. Sefton B.M. Anal. Biochem. 1989; 176: 22-27Crossref PubMed Scopus (325) Google Scholar). NIH-3T3 cells were transfected using the calcium phosphate method using 5 μg of DNA. After transfection, the cells were replated on glass coverslips contained in a 6-well tissue culture dish and allowed to grow for 36 h after which the cells were starved in Dulbecco's modified Eagle's medium and 0.1% calf serum for 16 h. Cells were activated with the appropriate compounds and then fixed with 3% paraformaldehyde for 20 min. After one wash with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min and blocked in 3% bovine serum albumin in PBS for 30 min. The primary monoclonal antibody, anti-HA antibody (antibody unit, WIS) was applied to the cells at a final concentration of 1 μg/ml in PBS for 45 min, washed with PBS, and incubated again with lissamine rhodamine-conjugated goat anti-mouse IgG and IgM (Jackson ImmunoResearch Laboratories) 1:200 in PBS for 45 min. After washing with PBS, coverslips were mounted, analyzed, and photographed using a Zeiss Axiophot camera. The assay was performed as described by Spengler et al. (12Spengler D. Waeber C. Pantaloni C. Holsboer F. Bockaert J. Seeburg P.H. Journot L. Nature. 1993; 365: 170-175Crossref PubMed Scopus (1123) Google Scholar). Briefly, Madin-Darby canine kidney cells expressing a stable luciferase construct containing of the were transfected by with an expression vector containing h after transfection, serum was from the cells, and after a of h, were h after stimulation, the medium was and the cells were washed with The cells were then lysed by adding luciferase buffer were transferred to the and luciferase activity was The were analyzed using Corp.). cpg16 was (2Hevroni D. Rattner A. Bundman M. Lederfein D. Gbarah A. Mangelus M. Silverman M.A. Kedar H. Naor C. Kornuc M. Hanoch T. Seger R. Theill L.E. Nedivi E. Richter-Levin G. Citri Y. J. Mol. Neurosci. 1998; 10: 75-98Crossref PubMed Scopus (138) Google Scholar) in a cDNA from rat hippocampus performed Northern of from rat rat rat and kidney with a probe of the 1600-base pair cDNA of cpg16 as described was in the rat brain and rat with of the cpg16 after with In of the other expression of cpg16 was a be in the kidney a not was in a the expression of this gene may not be to the and it a in The open reading frame of cpg16 a putative protein of approximately 50 on the primary of CPG16 a protein serine/threonine kinase containing of T. Methods Enzymol. 1991; PubMed Scopus Google Scholar). To CPG16 is a protein kinase and to its cpg16 was in-frame with cDNA of GST, expressed in and purified using glutathione of the purified revealed one protein at which to the for the GST-CPG16 protein. that be in the purified at the of is the protein In the of and the purified autophosphorylation in a that the cpg16 cDNA for a kinase A that was upon autophosphorylation a of the GST-CPG16 as by its when were from the buffer in the of not To for studies of CPG16 in cells, the cDNA was into a expression vector containing an HA The was transfected into COS7 cells by of the cells, and of the was when the purified was to with the anti-HA antibody which to the of CPG16 to a with an was upon of the purified proteins with the is the CPG16 with both HA and showed low autophosphorylation activity. a a possible for CPG16, we performed an in vitro substrate assay in which we a of and for to be by and ATP as CPG16 to have a as it showed activity only at a low of and This of CPG16 was when brain which of was used as a In of the CPG16 failed to phosphorylate any of the proteins in the in of the a be upon phosphorylation with CPG16 not of the GST-CPG16 and of revealed that the phosphorylation only on and This is with the protein serine/threonine kinase of cpg16 (2Hevroni D. Rattner A. Bundman M. Lederfein D. Gbarah A. Mangelus M. Silverman M.A. Kedar H. Naor C. Kornuc M. Hanoch T. Seger R. Theill L.E. Nedivi E. Richter-Levin G. Citri Y. J. Mol. Neurosci. 1998; 10: 75-98Crossref PubMed Scopus (138) Google Scholar, T. Methods Enzymol. 1991; PubMed Scopus Google for basic protein Jun N-terminal kinase MAPK/ERK kinase light chain receptor vitro phosphorylation assays were performed as described in a In vitro phosphorylation assays were performed as described with other protein CPG16 the to of the Ca2+/calmodulin-dependent protein and in to Ca2+/calmodulin-dependent protein kinase was to be a in plasticity M. J. Cell. Full Text PDF PubMed Scopus Google Scholar, K. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), by the transcription factor CREB H. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). and are capable of the autophosphorylation activity of that and the kinase EGTA, and had any effect on the autophosphorylation of the expressed GST-CPG16 not was effect of on GST-CPG16 activity any of the substrates in in vitro phosphorylation assays not Moreover, when the of cpg16 was with the of CaMKII, the of from the in CPG16 and a GST-CPG16 did not to a any not suggesting that CPG16 is not on or for its direct activity. the activity of many protein serine/threonine be in response to extracellular we to the low activity of CPG16 be by any extracellular Thus, COS7 cells were transfected with and treated with agents that signaling HA-CPG16 was then using anti-HA and to in vitro kinase assays. both forskolin and which the of a of HA-CPG16 purified from cells a in its autophosphorylation which at 30 min after and again to min after stimulation and cAMP 5 and not the of in response to was and at 45 min after The of isobutylmethylxanthine to inhibit and cAMP had only a effect on the of the phosphorylation of extracellular agents that were used to or of HA-CPG16 included the the protein kinase the inhibitor vanadate, epidermal growth and compounds are to downstream signaling and but had effect on the autophosphorylation activity of CPG16 5 and not Moreover, after from stimulated cells, was of CPG16 activity any of the in vitro substrates not the performed on COS7 cells, it that CPG16 is activated by cAMP-elevating which the of the protein kinase we used the PKA inhibitor to PKA is involved in CPG16 Thus, was added to the cells before stimulation by forskolin or H. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and the autophosphorylation activity of CPG16 was as described show that significantly inhibited the forskolin stimulation of CPG16 when added to the transfected COS7 cells min before stimulation the involvement of PKA in the of then CPG16 be a direct substrate of either PKA or that was to phosphorylate a in the of CPG16 J. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, R. Krebs E.G. J. PubMed Scopus Google Scholar). Although both the of PKA and activated were active in vitronectin and MBP, phosphorylation of purified HA-CPG16 by be after a phosphorylation reaction Recombinant active MAPK/ERK kinase (7Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar) and kinase failed to phosphorylate HA-CPG16 not and CPG16 had effect on kinases. the of CPG16 may by a cAMP-dependent a kinase to the of kinase downstream of PKA D. 1997; PubMed Google Scholar). To on the localization of CPG16, we performed studies in COS7 and in NIH-3T3 cells transfected with In both of the HA was in the in nuclear be after and forskolin stimulation of the cells and not This nuclear not be when the cells were stimulated with FCS in which are to the (7Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). Although mostly the that CPG16 may have a nuclear upon cAMP The of CPG16 to the the that it in the regulation of transcription. the phosphorylation of CPG16 is by we the that overexpression of CPG16 gene expression the transcription factor Madin-Darby canine kidney cells expressing a stable luciferase construct containing of the were stimulated with various cAMP-elevating and luciferase activity was (12Spengler D. Waeber C. Pantaloni C. Holsboer F. Bockaert J. Seeburg P.H. Journot L. Nature. 1993; 365: 170-175Crossref PubMed Scopus (1123) Google Scholar). of cells with 8-Br-cAMP, epidermal growth and but not calcium and phorbol the activity of the luciferase gene when cells were transfected with a of expression vector containing the cDNA for CPG16 a with the vector was in the luciferase activity of the vector containing the CPG16 did not have a effect on this activity. Thus, it is possible that CPG16 participates in the down-regulation of cAMP-induced the of may that this be an long term plasticity changes in central nervous system at in by of transcription Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, T. 1994; PubMed Scopus Google Scholar). transcription a of gene the many of which transcription for M. 1990; Full Text PDF PubMed Scopus Google Scholar). The gene in of downstream many of which are to changes in brain and are as candidate plasticity-related genes (CPGs (1Nedivi E. Hevroni D. Naot D. Israeli D. Citri Y. Nature. 1993; 363: 718-722Crossref PubMed Scopus (437) Google Scholar)). Hevroni et al. (2Hevroni D. Rattner A. Bundman M. Lederfein D. Gbarah A. Mangelus M. Silverman M.A. Kedar H. Naor C. Kornuc M. Hanoch T. Seger R. Theill L.E. Nedivi E. Richter-Levin G. Citri Y. J. Mol. Neurosci. 1998; 10: 75-98Crossref PubMed Scopus (138) Google Scholar) genes that are either or upon of the genes was CPG16, which with the plasticity-related In this we to or not CPG16 is a Ca2+/calmodulin-dependent kinase. A of the of CPG16 revealed that for are we that CPG16 failed to to a that the was not to Moreover, effect of or on the in vitro or in activity of CPG16 be that CPG16 is by a that involved in the of kinases. Although not by and a was when COS7 cells were transfected with stimuli that were for to CPG16 activity cAMP-elevating agents were to have a Thus, forskolin or CPG16 autophosphorylation activity by its other stimuli did not have any The which PKA the cAMP-dependent stimulation, that CPG16 downstream of CPG16 not be or activated by PKA in Moreover, the was the of PKA which may that CPG16 may be activated by a that be a kinase cascade. of phosphorylation by an kinase is of the of in the of CPG16 that is to the of many activated protein as ERK and MAPK/ERK kinase (8Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar). at this the direct of is not The relatively of be by to as be a possible on or that may be to but are not expressed in in the COS7 is possible that the the substrate of CPG16 is the of its autophosphorylation or activity The involvement of cAMP in neuronal plasticity is well and many studies have the involvement of this in the plasticity of memory and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cAMP is to its PKA that may either in the or to the and processes 1997; PubMed Scopus Google Scholar). In the the may as well as protein processes 1997; PubMed Scopus Google Scholar). the of PKA in the is to the transcription of cAMP-dependent which is by phosphorylation of the transcription factor CREB H. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In this the of CPG16 by and its by cAMP may that this kinase is one of the that participates in plasticity overexpression of the of PKA, the overexpression of CPG16 seems to inhibit the cAMP-stimulated CREB activity. the effect of PKA, CPG16 is to in the down-regulation of cAMP in the processes have been in of the PKA the down-regulation of by of cAMP by or of PKA protein activity has been to activity T. S. R. 1998; PubMed Scopus Google Scholar). CPG16 may in the regulation of CREB either by its or via the of that are involved in the of Although the of CPG16 in the is it be that its localization is and only a of nuclear was upon of CPG16 and the activity of other downstream in the have to be a possible substrate for CPG16 from the of T. K. D. M. N. N. A. H. N. O. DNA 1997; PubMed Scopus Google Scholar). This cDNA was to have a of to both CPG16 and A. A. A. E. J. Y. M. A. C. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. I. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), the is to the and is possible that CPG16 is involved in the regulation of or to one of its is to this In we have characterized a neuronal plasticity-related protein kinase, Although it to protein we have that it is not by or but is activated by cAMP-elevating CPG16 is not an in for PKA, suggesting that it may be activated by a cascade. CPG16 was to into the upon stimulation and to have a effect on cAMP-stimulated activity of CPG16 may in the down-regulation of cAMP plasticity-related and Nedivi for and on the as well as Hanoch for and and for with the assays.