Phosphoinositide-dependent Kinase Phosphorylation of Protein Kinase C Apl II Increases during Intermediate Facilitation inAplysia

Phosphorylation of protein kinase Cs (PKCs) by phosphoinositide-dependent kinase I (PDK) is critical for PKC activity. In the nervous system of the marine molluskAplysia, there are only two major PKC isoforms, the calcium-activated PKC Apl I and the calcium-independent PKC Apl II, and both PKCs are persistently activated during intermediate memory. We monitored the PDK-dependent phosphorylation of PKC Apl I and PKC Apl II using phosphopeptide antibodies. During persistent activation of PKCs in Aplysia neurons, there is a significant increase in the amount of PDK-phosphorylated PKC Apl II in the particulate fraction but no increase in the amount of PKC Apl I phosphorylated by PDK. PDK phosphorylation of PKCs was not sensitive to inhibitors of phosphatidylinositol 3-kinase, PKC, or expression of a kinase-inactive PDK. Localization of PDK-phosphorylated PKC Apl II using immunocytochemistry revealed an enrichment of phosphorylated PKC Apl II at the plasma membrane. These data suggest that increased PDK phosphorylation of PKC Apl II is important for persistent kinase activation. Phosphorylation of protein kinase Cs (PKCs) by phosphoinositide-dependent kinase I (PDK) is critical for PKC activity. In the nervous system of the marine molluskAplysia, there are only two major PKC isoforms, the calcium-activated PKC Apl I and the calcium-independent PKC Apl II, and both PKCs are persistently activated during intermediate memory. We monitored the PDK-dependent phosphorylation of PKC Apl I and PKC Apl II using phosphopeptide antibodies. During persistent activation of PKCs in Aplysia neurons, there is a significant increase in the amount of PDK-phosphorylated PKC Apl II in the particulate fraction but no increase in the amount of PKC Apl I phosphorylated by PDK. PDK phosphorylation of PKCs was not sensitive to inhibitors of phosphatidylinositol 3-kinase, PKC, or expression of a kinase-inactive PDK. Localization of PDK-phosphorylated PKC Apl II using immunocytochemistry revealed an enrichment of phosphorylated PKC Apl II at the plasma membrane. These data suggest that increased PDK phosphorylation of PKC Apl II is important for persistent kinase activation. protein kinase C phosphoinositide-dependent protein kinase 4,5)P3, phosphatidylinositol 3,4,5-triphosphate serotonin or 5-hydroxytryptamine phosphate-buffered saline bisindolylmaleimide green fluorescent protein glutathione S-transferase maltose-binding protein pleckstrin homology phosphatidylinositol 3-kinase In the marine mollusk, Aplysia californica, PKCs1 are important for both short and intermediate term changes in synaptic strength between sensory and motor neurons that accompanies behavioral sensitization (1Byrne J.H. Kandel E.R. J. Neurosci. 1996; 16: 425-435Crossref PubMed Google Scholar). Following behavioral sensitization, or prolonged treatment of ganglia with serotonin (5-HT), the kinase activity in the particulate fraction of both Ca2+-activated conventional PKC Apl I and Ca2+-independent novel PKC Apl II is increased (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, 3Sossin W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). Persistent PKC activity is required for the maintenance of synaptic facilitation under some conditions (4Sutton M.A. Carew T.J. Neuron. 2000; 26: 219-231Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The mechanisms for the persistent activation of the PKCs are not well defined. Both initial PKC activation and protein translation are required for persistent activation of the PKCs (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar). However, there are differences in the persistent activation of the two isoforms. The increase in PKC Apl II activity appears to be mainly because of autonomous activation of the kinase, whereas the increase in PKC Apl I appears to be mainly due to increased levels of regulated PKC Apl I on the membrane (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, 3Sossin W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). There is increased phosphorylation of PKC Apl I at a conserved autophosphorylation site during intermediate memory; however, the phosphorylated kinase is located exclusively in the cytoplasm and presumably does not contribute to the increase in particulate kinase activity (5Nakhost A. Dyer J.R. Pepio A.M. Fan X. Sossin W.S. J. Biol. Chem. 1999; 274: 28944-28949Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Increased phosphorylation of PKC has been postulated to underlie persistent activation of PKCs during vertebrate learning models (6Sweatt J.D. Atkins C.M. Johnson J. English J.D. Roberson E.D. Chen S.J. Newton A. Klann E. J. Neurochem. 1998; 71: 1075-1085Crossref PubMed Scopus (51) Google Scholar, 7Atkins C.M. Selcher J.C. Petraitis J.J. Trzaskos J.M. Sweatt J.D. Nat. Neurosci. 1998; 1: 602-609Crossref PubMed Scopus (952) Google Scholar, 8Klann E. Chen S.J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar), and PKC activity is regulated by phosphorylation (9Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar). All PKCs require phosphorylation at the activation loop site by phosphoinositide-dependent kinase 1 (PDK) for full catalytic activity (10Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Ca2+-activated PKCs are phosphorylated by PDK soon after translation (11Dutil E.M. Newton A.C. J. Biol. Chem. 2000; 275: 10697-10701Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 13Sonnenburg E.D. Gao T. Newton A.C. J. Biol. Chem. 2001; 276: 45289-45297Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). After PDK-dependent phosphorylation, Ca2+-activated PKCs undergo two major autophosphorylations that are required for the stability and folding of the enzyme, a process termed maturation (9Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar). Once these sites are phosphorylated, the PDK site can become de-phosphorylated without affecting the stability of the enzyme (12Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Because PDK docks at a C-terminal site that is no longer easily accessible in the mature enzyme, classical PKCs are not easily re-phosphorylated (14Gao T. Toker A. Newton A.C. J. Biol. Chem. 2001; 276: 19588-19596Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Thus, whereas overexpression of PDK or PDK dominant-negatives affect the proportion of PKC found in the mature fraction, it does not affect the amount of mature PKC phosphorylated at the PDK site (12Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), presumably due to the lack of re-phosphorylation. PDK phosphorylation of novel PKCs has also been examined, although a detailed model for their phosphorylation has not been proposed. In contrast to Ca2+-activated PKCs (13Sonnenburg E.D. Gao T. Newton A.C. J. Biol. Chem. 2001; 276: 45289-45297Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), PDK phosphorylation of PKCδ is regulated both by PKC autophosphorylation and by PI 3-kinase activity (15Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). Regulation of phosphorylation of other kinases by PDK appears to be dependent on substrate conformation and subcellular localization as opposed to activation of PDK itself (16Belham C., Wu, S. Avruch J. Curr. Biol. 1999; 9: R93-R96Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 17Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 18Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). PDK has an N-terminal kinase domain and a C-terminal pleckstrin homology (PH) domain that binds phosphoinositides including phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) (19Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 20Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar, 21Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (916) Google Scholar), although kinase activity does not require PtdIns(3,4,5)P3 binding (22Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). We have cloned PDK from Aplysia, and both the kinase domain and the PH domain are highly homologous to both vertebrate and invertebrate PDKs, suggesting that this is a PDK orthologue (23Khan A. Pepio A.M. Sossin W.S. J. Neurosci. 2001; 21: 382-391Crossref PubMed Google Scholar). To examine the role of PDK in the regulation of PKC inAplysia, we raised phosphopeptide antibodies to the PDK site in the classical PKC Apl I and the novel PKC Apl II. We then used the antibodies to characterize PDK phosphorylation of PKCs in theAplysia nervous differences in PDK regulation of classical and novel PKCs in the nervous system and suggest an important role for PDK phosphorylation of PKC Apl II during intermediate A. from at and in an for at The in a of and then by of and ganglia from the and to in (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, M. Kandel E.R. H. J. Schwartz J.H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The ganglia then in to of and in (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, M. Kandel E.R. H. J. Schwartz J.H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google for at to of In some from ganglia and in a the conserved PDK site in PKC Apl I and Apl II with the to The to the and at In to the fraction of that the the from was an of the of the to process was After antibodies from the The was then an of the phosphopeptide to and antibodies and in a to PKC Apl PKC Apl II, and PDK have been (23Khan A. Pepio A.M. Sossin W.S. J. Neurosci. 2001; 21: 382-391Crossref PubMed Google Scholar, W.S. Schwartz J.H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of PDK PKC Apl PKC Apl II, and PKC Apl II (23Khan A. Pepio A.M. Sossin W.S. J. Neurosci. 2001; 21: 382-391Crossref PubMed Google Scholar, W.S. Fan F. J. Neurosci. 1996; 16: PubMed Google Scholar, F. Fan X. T. Sossin W.S. J. Neurosci. 2001; 21: PubMed Google used at a of of for and membrane was as F. Fan X. T. Sossin W.S. J. Neurosci. 2001; 21: PubMed Google Scholar). The and have been (23Khan A. Pepio A.M. Sossin W.S. J. Neurosci. 2001; 21: 382-391Crossref PubMed Google Scholar). We these in and the on glutathione The then with of with PKC Apl I or PKC Apl II. The and then with The then with and on in of nervous system (5Nakhost A. Dyer J.R. Pepio A.M. Fan X. Sossin W.S. J. Biol. Chem. 1999; 274: 28944-28949Abstract Full Text Full Text PDF PubMed Scopus (22) Google was for with and then with of the with The protein used to PKC Apl I was by a of PKC Apl I with The protein with the from the and the C-terminal The protein used to PKC Apl II was by a the catalytic from PKC Apl II with and The of this in with and to the that been with and the in with The of PKC Apl II with and the C-terminal The PDK and PDK used to have been (23Khan A. Pepio A.M. Sossin W.S. J. Neurosci. 2001; 21: 382-391Crossref PubMed Google Scholar). To an expression for Aplysia neurons we PDK from the with and and it the Apl II F. Fan X. T. Sossin W.S. J. Neurosci. 2001; 21: PubMed Google with the domain of PKC with the PDK to an protein with from Aplysia PDK. the of Aplysia this is not conserved and does not of the kinase or PH The for intermediate facilitation was to that (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, 3Sossin W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar, A. Dyer J.R. Pepio A.M. Fan X. Sossin W.S. J. Biol. Chem. 1999; 274: 28944-28949Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). kinase activity on the was as W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). as W.S. Schwartz J.H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). as J.R. Pepio A.M. Sossin W.S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google with the to phosphorylated PKC Apl II at 1 the to phosphorylated PKC Apl I at 1 the to PKC Apl I at and the to PKC Apl II at 1 The phosphopeptide antibodies with the at a of was used at by and was using the domain at the of and on the at We data with the of that the data using the is the that after this with to amount of protein a of with both and antibodies a was of with of although not between was for differences in between and or between the was to a was between and conditions or To the for was from a was To and autonomous for the for the or autonomous activity was to and other to the of on the The was to that F. Fan X. T. Sossin W.S. J. Neurosci. 2001; 21: PubMed Google with the on with to increase process for to expression with and then for in in with then in in with for and with with a in in binding was by with a in for 1 with the the PKC and in was The for with and then with in and for other the phosphopeptide was using the of the at 1 for to and then was to the in a in for in and then with in and for the for with and the using as the on a with All was on The of plasma membrane and Aplysia neurons have highly due to their and in a the plasma membrane is not well defined. We used a as a of plasma to for from the of for between be in the sensory that using the used for the PKC Apl II including these not used in the the was for and membrane for both and antibodies. was then for and and then the for was To examine the of with we the of the the membrane and the for the and on the to an of for The that is phosphorylated by PDK in vertebrate PKCs is well conserved in the Aplysia PKCs We raised antibodies to in this for both PKC Apl I and PKC Apl II. We the used for to the N-terminal of the PDK site in to antibodies that are for PKC and that not other PDK-phosphorylated The antibodies highly for PKC from nervous system that with the by well antibodies to PKC Apl I or PKC Apl II (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, W.S. Schwartz J.H. J. Biol. Chem. 1993; Full Text PDF PubMed Google To the of the antibodies for phosphorylated PKC, we PKC catalytic domain as a because there is no PDK in and the PKC not be phosphorylated at the PDK that the antibodies are highly for phosphorylated PKCs as not with PKC in 1 The by the phosphopeptide antibodies are due to to PKC as can be by antibodies to PKC Phosphorylation at the PDK site is not due to because a kinase-inactive PKC F. Fan X. T. Sossin W.S. J. Neurosci. 2001; 21: PubMed Google was well phosphorylated at the PDK site in However, the of phosphorylation at the PDK site in the kinase-inactive PKC was that for the kinase in 1 be due to increased of the kinase PKC Apl II. phosphorylation at the PDK site was sensitive to in the for the kinase-inactive PKC Apl II for PKC Apl II in 1 is with from classical PKCs that PKC autophosphorylation is required to a that the PDK site from (11Dutil E.M. Newton A.C. J. Biol. Chem. 2000; 275: 10697-10701Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In PDK binds to To PDK to PKC Apl II, we PKC Apl II was by the protein but was not by the PKC phosphorylated at the PDK site was not as well as PKC in have been for PDK de-phosphorylated PKC phosphorylated PKC (14Gao T. Toker A. Newton A.C. J. Biol. Chem. 2001; 276: 19588-19596Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). for this is that PDK binds to the C-terminal of PKCs and that this binding site is not accessible in the mature of the kinase (14Gao T. Toker A. Newton A.C. J. Biol. Chem. 2001; 276: 19588-19596Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Thus, a proportion of PKC Apl II that was be in an and to have C-terminal accessible for binding to PDK. we these using PKC Apl II from the nervous the proportion of phosphorylated and PKC Apl II to was in be due to the proportion of kinase in the mature nervous with PKC Apl II is in the The persistent activation of PKCs is not but increased phosphorylation of PKCs by PDK is We PDK phosphorylation of PKCs was during the persistent activation of the PKCs by prolonged treatment with the (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar). there was a significant increase in the of PKC Apl II phosphorylated at the PDK site in the fraction a treatment of Aplysia ganglia with and There was no in the of PKC Apl II phosphorylated at the PDK site or in the of or particulate PKC Apl I phosphorylated at the PDK site and (2Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar), there was a significant increase in the of PKC Apl I in the particulate fraction and there was no significant in the of PKC Apl II in the particulate fraction, the of PDK-phosphorylated PKC Apl II in the particulate fraction increase In PKC Apl II that was not phosphorylated by PDK was mainly in the fraction the of PDK phosphorylation was in the After however, there was a in this PDK phosphorylation in the C In PKC Apl I phosphorylation at the PDK site in and and after treatment C and kinase activity is also increased by during intermediate and this activity from PKC Apl II W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). In a of we both the of phosphorylation of PKC Apl II at the PDK site and autonomous kinase activity from the of ganglia with There was a between the levels of autonomous activity and the a between PDK phosphorylation and autonomous activity of PKC Apl II. To mechanisms that the of PDK to PKC in the Aplysia nervous we PDK phosphorylation of PKC was regulated by PKC activity or by PI 3-kinase activity as has been for Ca2+-independent PKCs in (15Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). inhibitors of PKC PI 3-kinase the of phosphorylation at the PDK site of PKC phosphorylation to an increased of PKC on the membrane for both PKC Apl I and PKC Apl II with PKC activity required for the of PKC from the particulate fraction (5Nakhost A. Dyer J.R. Pepio A.M. Fan X. Sossin W.S. J. Biol. Chem. 1999; 274: 28944-28949Abstract Full Text Full Text PDF PubMed Scopus (22) Google X. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and We also the activity and amount of PKC Apl II on of neurons in a W.S. Chen Toker A. J. Neurochem. 1996; PubMed Scopus (23) Google Scholar), to in PDK phosphorylation of PKC Apl II. significant changes in the levels of PKC Apl II phosphorylated at the PDK site after treatment in of fraction by The antibodies to phosphorylated PKC also well in immunocytochemistry of Aplysia To the localization of phosphorylated and PKC, we raised an to PKC Apl II in as the raised to the and was in immunocytochemistry and using the we to PKC Apl II phosphorylated at the PDK site and PKC Apl II in the the two revealed a significant increase in the of Apl II on or the plasma membrane of C in between the antibodies was in or and To we phosphorylation of PKC by using a we the of for and PKC in neurons that with a a kinase-inactive PDK. The PDK was with to of There was no significant in the of PDK-phosphorylated PKC Apl II to PKC Apl II in the cytoplasm or between or not the PDK Thus, at the levels of expression we can in neurons, kinase-inactive PDK does not to as a to PKC phosphorylation by PDK. We phosphopeptide antibodies to the conserved PDK site in PKC Apl I and PKC Apl II. These antibodies are for PKCs phosphorylated at the PDK site as not PKCs in no activity is the by the phosphopeptide antibodies can be by other antibodies to The major from this is that phosphorylation at the PDK site of PKC Apl II is increased after prolonged treatment and is with autonomous PKC Apl II activity. In the increased activation of PKC Apl I at this is not with increased phosphorylation at the PDK We also PDK-phosphorylated PKC Apl II using immunocytochemistry and an enrichment of phosphorylated PKC Apl II at or the plasma membrane. Phosphorylation of Ca2+-independent or novel PKCs at the PDK site appears to be regulated that of conventional phosphorylation of the novel PKCδ by PDK PtdIns(3,4,5)P3 and (15Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar), whereas in phosphorylation of Ca2+-activated PKCs does not (12Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). phosphorylation of PKCδ in of conventional PKCs because PKCδ is in the PDK site is not phosphorylated (15Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). Thus, PKCδ can in the of PDK phosphorylation D.B. Ziegler Parker P.J. J. 2000; PubMed Scopus Google Scholar). PKC Apl II however, there has been of phosphorylation by PDK. In contrast to there is a of PKC Apl II phosphorylation at the PDK and this was not sensitive to PI 3-kinase inhibitors or PKC Thus, it is that phosphorylation of PKC Apl II at the PDK site the phosphorylation of conventional PKCs (15Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). important is that we are PKC in nervous system there is activation of during the of we of the Thus, that of phosphorylation of PKC at the PDK site does not require PI 3-kinase or PKC but not the initial phosphorylation of PKC Apl II at the PDK site is regulated in the nervous PDK phosphorylation of PKC Apl II does to be regulated PDK phosphorylation of PKC Apl PKC Apl II that is not phosphorylated at the PDK site has a subcellular using it is in the fraction and by using it is from the plasma membrane These suggest that of PKC Apl II at the PDK site PKC localization or that a of PKC Apl II is not phosphorylated at the PDK site during appears from PKC Apl I PKC Apl I is not found in the on phosphorylation at the PDK site there is increased phosphorylation of PKC Apl II but not PKC Apl I in the fraction during intermediate facilitation There was no significant in levels of PKC Apl II in the suggesting that this increase was due to increased phosphorylation of the PDK The mechanisms that this phosphorylation are a of particulate PKC Apl II is at this W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar), and phosphorylation at the PDK site be by the of a in the is by the between autonomous kinase activity and the of PKC phosphorylated at the PDK site this it is not the increased PDK phosphorylation is important in autonomous activation of the kinase, or a of the autonomous activation. because the PDK not as a and because there are no inhibitors of we at this PDK is required for activation of PKC during intermediate using immunocytochemistry we found that PKC Apl II phosphorylated at the PDK site is or on the plasma membrane. that the of PKC is from the plasma membrane. is with that there is a of particulate PKC that is not phosphorylated The particulate of PKC not phosphorylated at the PDK site be with the or an membrane fraction and be in as a of particulate PKC Apl II in Aplysia is A. P. Sossin W.S. J. Neurochem. 1998; 71: PubMed Scopus Google Scholar). PDK phosphorylation of PKCs has been well in and in this is the of PDK phosphorylation in There important differences in the PDK phosphorylation of the classical PKC Apl I and the novel PKC Apl II These differences be by that classical PKCs are only phosphorylated at the PDK site during initial of the kinase, whereas novel PKCs be regulated by of this site during the kinase of classical PKCs that to of the kinase G. F. Hemmings B.A. Parker P.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In novel PKCs be de-phosphorylated and as an of enzyme that can be at a novel PKCs are by in J. Neurosci. 1998; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar, K. S. M. Sacktor T. H. J. Neurosci. 2000; PubMed Google Scholar, J. Neurosci. 1997; PubMed Scopus Google Scholar, A. S. M. F. Neurosci. 1996; PubMed Scopus Google Scholar), including Aplysia neurons W.S. Schwartz J.H. 1994; PubMed Scopus Google is also the PDK phosphorylation has been by The increase in at for PKC Apl II phosphorylated at the PDK site be with increased PDK phosphorylation of activated PDK phosphorylation of PKC Apl II a role in activation during intermediate The activation of PKC Apl II at this translation W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). PDK phosphorylation to the important translation to be regulated to persistently PKC Apl II during intermediate We and for on the and Fan for

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