Protein Kinase C Phosphorylated at a Conserved Threonine Is Retained in the Cytoplasm

Abstract

Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 inAplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation. Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 inAplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation. protein kinase C phosphatidylserine 5-hydroxytryptamine (serotonin) phorbol dibutyrate phorbol 12-myristate 13-acetate polyacrylamide gel electrophoresis polymerase chain reaction Protein kinase Cs (PKCs)1 represent a large family of proteins that undergo complex regulation. Classical isoforms of PKC are activated by a combination of two second messengers, calcium and diacylglycerol. Calcium increases the affinity of the enzyme for lipids through its interaction with the C2 domain, and diacylglycerol induces a high affinity interaction with the membrane that leads to enzyme activation (1Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1471) Google Scholar, 2Newton A.C. Curr. Biol. 1995; 5: 973-976Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In order to be activable by second messengers, PKC is first activated through phosphorylation by both phosphoinositide-dependent kinase 1 (3Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (973) Google Scholar, 4Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 5Chou M.M. Hou W.M. Johnson J. Graham L.K. Lee M.H. Chen C.S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar) and by autophosphorylation (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Two major autophosphorylation sites have been identified in classical PKCs. Autophosphorylation at threonine 641 (PKC βII) is required for formation of active PKC (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 7Dutil E.M. Keranen L.M. DePaoli-Roach A.A. Newton A.C. J. Biol. Chem. 1994; 269: 29359-29362Abstract Full Text PDF PubMed Google Scholar, 8Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google Scholar, 9Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 10Edwards A.S. Faux M.C. Scott J.D. Newton A.C. J. Biol. Chem. 1999; 274: 6461-6468Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and autophosphorylation at serine 660 (PKC βII) is important for stabilization of enzyme conformation (11Bornancin F. Parker P.J. J. Biol. Chem. 1997; 272: 3544-3549Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 12Edwards A.S. Newton A.C. J. Biol. Chem. 1997; 272: 18382-18390Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Interestingly, mutations at serine 660 also affect the affinity of the enzyme for calcium, suggesting that in the intact kinase interactions occur between the C2 domain and the carboxyl terminus (12Edwards A.S. Newton A.C. J. Biol. Chem. 1997; 272: 18382-18390Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). A number of other autophosphorylation sites have been identified after activation of PKCs by phorbol esters (13Flint A.J. Paladini R.D. Koshland D.J. Science. 1990; 249: 408-411Crossref PubMed Scopus (108) Google Scholar), but these sites are not important for PKC activity (8Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google Scholar, 14Zhang J. Wang L. Petrin J. Bishop W.R. Bond R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6130-6134Crossref PubMed Scopus (33) Google Scholar). However, one of these sites, threonine 634 in PKC βII, is conserved over evolution. Furthermore, phosphorylation at threonine 634 and threonine 641 increases after long term potentiation or after associative learning in rodents (15Atkins C.M. Selcher J.C. Petraitis J.J. Trzaskos J.M. Sweatt J.D. Nat. Neurosci. 1998; 1: 602-609Crossref PubMed Scopus (949) Google Scholar, 16Sweatt 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), suggesting a role for phosphorylation at these sites in regulation of PKC in the nervous system. In the marine mollusk Aplysia californica, only two phorbol ester-activated isoforms of PKC exist, PKC Apl I and PKC Apl II (17Sossin W.S. Diaz-Arrista R. Schwartz J.H. J. Biol. Chem. 1993; 268: 5763-5768Abstract Full Text PDF PubMed Google Scholar). PKC Apl I is a classical PKC that contains a C2 domain that is regulated by calcium, while PKC Apl II contains an amino-terminal C2 domain that is homologous to those of PKCε and PKCη and not regulated by calcium (18Pepio A.M. Fan X. Sossin W.S. J. Biol. Chem. 1998; 273: 19040-19048Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The major autophosphorylation sites in vertebrate PKCs are conserved in the Aplysia isoforms, as is the site at threonine 634 (Fig. 1). PKC is persistently activated during the formation of long term synaptic changes in both vertebrates and invertebrates (19Grunbaum L. Muller U. J. Neurosci. 1998; 18: 4384-4392Crossref PubMed Google Scholar, 20Klann E. Chen S.J. Sweatt J.D. J. Biol. Chem. 1991; 266: 24253-24256Abstract Full Text PDF PubMed Google Scholar, 21Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, 22Wang J.H. Feng D.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2576-2580Crossref PubMed Scopus (149) Google Scholar, 23Farley J. Schuman E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2016-2020Crossref PubMed Scopus (46) Google Scholar). In both model systems, an autonomous form of PKC is formed that may be important for the maintenance of synaptic changes at intermediate times (19Grunbaum L. Muller U. J. Neurosci. 1998; 18: 4384-4392Crossref PubMed Google Scholar, 20Klann E. Chen S.J. Sweatt J.D. J. Biol. Chem. 1991; 266: 24253-24256Abstract Full Text PDF PubMed Google Scholar, 24Sossin W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). The autonomous form of PKC in vertebrates has been suggested to be due to phosphorylation of a classical isoform or the formation of protein kinase M from the atypical ζ form (25Klann E. Chen S.J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (156) Google Scholar, 26Sacktor T.C. Osten P. Valsamis H. Jiang X. Naik M.U. Sublette E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8342-8346Crossref PubMed Scopus (344) Google Scholar). In contrast, the autonomous PKC in Aplysia is generated by a modification in the regulatory domain of the novel PKC Apl II (24Sossin W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). Translocation of PKC Apl I to the membrane and the amount of activable PKC Apl I in the membrane also increase at this time, demonstrating that PKC Apl I is persistently activated during this period although not constitutively active (21Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar). To determine if phosphorylation of PKC Apl I at the equivalent positions of threonine 634 and threonine 641 (threonine 613 and threonine 620) is involved in the persistent activation of PKC Apl I, we raised a phosphopeptide antibody to this site. This antibody only recognized PKC Apl I when phosphorylated at threonine 613. We found that this site is phosphorylated in neurons and that the percentage of kinase phosphorylated at this site increased after phorbol ester and serotonin treatment. Interestingly, PKC Apl I phosphorylated at this site was found in the cytoplasmic fraction to a greater extent, and was more resistant to down-regulation, than the bulk of PKC. These results suggest that phosphorylation of this site is involved in removing PKC from the membrane. A 17-amino acid carboxyl-terminal peptide, containing residues 606–623 ofAplysia PKC Apl I with threonine 613 and threonine 620 converted to phosphothreonine (pT), was synthesized (FDREFpTSEAPNVTpTPTD; Fig. 1) at the Baylor College of Medicine protein chemistry core facility, conjugated to maleimide-coupled bovine serum albumin (Pierce), and injected into rabbits using the adjuvant TitreMax Gold (Cedar Lane) three times at 4-week intervals. In order to reduce the fraction of antibody that might recognize the nonphosphorylated peptide, the serum from animals was passed three times over an affinity column of the nonphosphorylated form of the immunizing phosphopeptide (synthesized at Sheldon Biotechnology Center, McGill University) coupled to SulfoLink (Pierce). After each passage, specifically retained antibodies were eluted from the column. The final flow-through was then passed over an affinity column of the immunizing peptide coupled to SulfoLink, and retained antibodies were eluted and concentrated in a Centriplus-10 (Amicon). Western blots were performed as described (27Dyer J.R. Sossin W.S. Klein M. J. Neurochem. 1996; 67: 932-942Crossref PubMed Scopus (21) Google Scholar) with the PKC Apl I-specific antibody at 0.2–1 μg/ml dilution (17Sossin W.S. Diaz-Arrista R. Schwartz J.H. J. Biol. Chem. 1993; 268: 5763-5768Abstract Full Text PDF PubMed Google Scholar), the antibody to the phosphorylated peptide (anti-Apl I-P) at 0.5 μg/ml, and goat anti-rabbit, horseradish peroxidase-conjugated secondary antibody at 1 μg/ml. For immunoblots with the phosphopeptide-specific antibody, nonphosphorylated peptide at 5 μg/μl was incubated with equal of the antibody for to its at 5 and its to the were by were and was performed using the domain at the of and the We with the of the using the is the that after this were with to the amount of protein over a of A. P. Sossin W.S. Neurochem. 1998; 71: PubMed Scopus Google Scholar). acid mutations were generated with a using the polymerase chain reaction For the first the Apl I in W.S. Fan F. J. Neurosci. 1996; PubMed Google Scholar) as a and the and the or the and as the The from the first were and as the for second using the two The was and and into the Apl I in the W.S. Fan F. J. Neurosci. 1996; PubMed Google Scholar) with the site was formed by the and was to the For the an site in PKC Apl I was for a using the and the The was and and into the Apl I in the W.S. Fan F. J. Neurosci. 1996; PubMed Google Scholar) with the A site was formed by the and was to the For the the were as for the with the and The was then with and and into the PKC Apl I with the A site was formed by the and was to the The was over the to changes were of the into and of high were performed as described W.S. Fan F. J. Neurosci. 1996; PubMed Google Scholar). Phosphorylation was by the of to the phosphorylation 5 μg/ml when of was were incubated in a These were to at for and were by the of of and then After to membranes were with by of the membrane and with the I The amount of autophosphorylation was using a were after in as described W.S. Fan F. J. Neurosci. 1996; PubMed Google Scholar). of PKCs using or were as described W.S. Fan F. J. Neurosci. 1996; PubMed Google Scholar). and were as described (18Pepio A.M. Fan X. Sossin W.S. J. Biol. Chem. 1998; 273: 19040-19048Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, A.C. Koshland J. Biol. Chem. Full Text PDF PubMed Google Scholar). For kinase using of 5 of were with of of and incubated for of were then and in of 1 5 and were performed at were for with a and and was by in a at for at A fraction of the was from while the was then and in PKC activity as described W.S. Fan F. J. Neurosci. 1996; PubMed Google Scholar). Aplysia were in an for at The animals were first in a of and then by of were from the and to in containing high and calcium (21Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar). The two and from each were and for in at (21Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar) with and from each was with while the other for 1 or for at In using both were then in three times and incubated in for at the of the the was with and the were and to containing of as in to 5 and 1 The were in the and at at for these were at for at The was and the were in of A from each was for of the was in and equal of protein from and were After to membranes were with by of the membrane and Western with We generated antibody to a phosphopeptide from PKC Apl I phosphorylated at both threonine 613 in PKC βII) and threonine 620 in PKC βII) We first antibody that to the nonphosphorylated peptide using an affinity column generated from a nonphosphorylated of the We then antibody that to an affinity column from the phosphorylated antibodies were from two but in this antibody from (anti-Apl To to phosphorylated the antibody was also incubated with nonphosphorylated peptide to these not recognize PKC Apl I in using of PKC Apl I with PKC and to the phosphorylation of Apl I and the of with These results suggest that the phosphorylation of threonine PKCs from are to be phosphorylated at threonine 620 activation (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, A.J. Koshland J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), but the PKC is only recognized by after with PKC and in To that the increase in was not due phosphorylation at threonine we both this threonine and the constitutively phosphorylated serine with acid (PKC Apl I The kinase was from with a the to PKC Apl I, this kinase was only by after with PKC and (Fig. a negative at threonine 620 was not for by but after phosphorylation of threonine 613 in were recognized by in PKC Apl I These may represent phosphorylated of PKC Apl I, phosphopeptide of PKC Apl I that autophosphorylation sites are in PKC Apl I from threonine threonine and serine not To that phosphorylation at threonine 613 was required for we phosphorylation of PKC Apl I after threonine 613 with alanine (PKC Apl I This was not with or after in the high activity of this protein (Fig. and its ability to at other positions (Fig. These results that recognizes PKC when it is phosphorylated at threonine 613. from vertebrate PKCs that the enzyme is phosphorylated at threonine 620 (8Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and that this site is phosphorylated in (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google A.J. Koshland J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). we that kinase phosphorylated at threonine 613 also be phosphorylated at threonine However, results not recognize PKC phosphorylated at threonine 613 a negative at threonine Phosphorylation of PKC Apl I at threonine 613 is a period of activation This is to the bulk of PKC Apl I autophosphorylation by of 613 is not the only site phosphorylated these of into PKC Apl I was with that of PKC Apl I (Fig. the of threonine 613 phosphorylation in this To determine if threonine 613 is phosphorylated in we Aplysia nervous that were for 1 with or 1 with of to the translocation of PKC Apl I, by W.S. Schwartz J.H. 1994; PubMed Scopus Google Scholar) a amount of translocation and due to the in the of the phorbol esters into the W.S. Schwartz J.H. 1994; PubMed Scopus Google Scholar). Two are in Fig. 1 a large amount of translocation with down-regulation, while in also with of the PKC phosphorylated at this in nervous systems, a pool of PKC Apl I was suggesting in to phosphorylation of threonine 613 occurs in neurons (Fig. This is not due to with PKC Apl I, of PKC Apl I the gel not although the amount of PKC Apl I the amount of kinase in the (Fig. the PKC phosphorylated at threonine 613 was found in the after the of PKC Apl I been to the membrane with (Fig. 1). This was active PKC is found in the and PKC have to be activated to the phosphorylation at threonine 613. phorbol esters increased the percentage of PKC Apl I phosphorylated at threonine 613 by This an increase in threonine PKC with a in PKC Apl I that is not phosphorylated at threonine due to down-regulation. with this the increase in the percentage of kinase phosphorylated at threonine 613 with the amount of PKC Apl I (Fig. phosphorylation at threonine 613 may PKC Apl I from down-regulation. This be due to the of may be by membrane A. M. Biol. 1998; 18: PubMed Scopus Google Scholar, L. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We also with the to an increase in PKC Apl I A increase in the percentage of PKC Apl I phosphorylated at threonine 613 was after a of (Fig. In this we not of PKC Apl I increased phosphorylation at threonine 613 occurs during the persistent activation of PKC in Aplysia to results during persistent activation of PKC in vertebrate 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), although in the vertebrate increased phosphorylation at other sites or an increase in PKC may also have to the 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). To determine the of threonine 613 for activation and translocation of we the for kinase activity of the PKC Apl I PKC Apl I as PKC in the of calcium. In contrast, PKC Apl I required calcium at for activation (Fig. These results suggest that this site may the of calcium to the C2 domain of PKC Apl it has been that phosphorylation of serine 660 is involved in the of calcium (12Edwards A.S. Newton A.C. J. Biol. Chem. 1997; 272: 18382-18390Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), but this is the first that phosphorylation at threonine 613 (threonine 634 in PKC βII) is also important for the interaction of calcium with the C2 This the that PKC has a affinity for calcium and a affinity for the membrane. We have to this by PKC Apl I in by translocation to the membrane. However, the for phosphorylation in to translocation to the membrane that is not not 634 (PKC βII) was identified as a major site of phosphorylation after activation of PKC (13Flint A.J. Paladini R.D. Koshland D.J. Science. 1990; 249: 408-411Crossref PubMed Scopus (108) Google phosphorylation of this site was in PKC in or after in (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, A.J. Koshland J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). with these we phosphorylation of the homologous site in PKC Apl I, threonine after in However, we have phosphorylation of this site in Aplysia neurons. This may be due to a greater of PKC activation in the nervous as with or with a antibody is also in although in this may be due to phosphorylation of threonine 641 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). 613 is conserved in classical PKCs and novel PKCs (Fig. 1). results that phosphorylation at this site a pool of enzyme that is retained in the after with phorbol esters and that resistant to down-regulation. results not that phosphorylation at threonine 613 is for in the other sites may to be phosphorylated in with threonine 613. Furthermore, we not Apl recognizes PKC phosphorylated at threonine 613 in the of phosphorylation at threonine it is that is a pool of PKC phosphorylated at but not at that is found the membrane. with the vertebrate PKC suggest that threonine 620 is constitutively phosphorylated (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, A.J. Koshland J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), this has not been for isoforms or in the nervous in results are with that a phosphorylated pool of PKC in is not to membranes by phorbol esters and is not A. P.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). is that this cytoplasmic pool of PKC to kinase phosphorylated at the homologous acid (threonine in PKC (Fig. 1). In this the phosphorylation only with activation by phorbol esters and not by calcium However, we a phosphorylation of threonine 613 in PKC Apl I after by and calcium or and in not is also in with the of Feng and X. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), that PKC autophosphorylation to be from In this of threonine 641 and serine 660 to alanine the ability of PKC to be from the membrane. However, this alanine was from the membrane with PKC. These results are with phosphorylation at threonine 634 (threonine 613 in for removal from the phosphorylation at threonine 641 and serine 660 may be important for the ability of PKC to at this (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). We an increase in phosphorylation of threonine 613 after to a is a persistent increase in PKC Apl I activity and autonomous PKC activity (21Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar). We not that phosphorylation at threonine 613 is important for persistent activation of PKC Apl I or formation of the autonomous threonine PKC is in the and is increase in the amount of activable PKC Apl I or in autonomous activity at this (21Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar). Furthermore, autonomous activity from the be with the antibody to PKC Apl I (21Sossin W.S. Sacktor T.C. Schwartz J.H. Learn. Mem. 1994; 1: 189-202PubMed Google Scholar, 24Sossin W.S. Learn. Mem. 1997; 3: 389-401Crossref PubMed Scopus (23) Google Scholar). We a model increased phosphorylation at threonine 613 is a of persistent activation of PKC but not a of persistent activation of PKC. A number of of PKC regulation have been to the phosphorylation of the and these phosphorylation to an increase in PKC activity (6Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). In contrast, we suggest that phosphorylation at threonine 613 is not important for kinase activity and may to PKC activity by PKC from with the membrane. This of PKC translocation be in The be due to a in the affinity for although we have not been to a in not may be a in the affinity of the enzyme for calcium, and translocation of PKC Apl I by phorbol esters calcium in Sossin W.S. Sacktor T.C. P.J. S. Schwartz J.H. J. Neurosci. 1991; PubMed Google Scholar). we the of translocation with in proteins may be required to PKC Apl I translocation in neurons. In a protein might specifically to PKC phosphorylated at threonine 613 and it in the the mechanism for of PKC the of translocation may to a in activable PKC. We suggest that phosphorylation of this is a mechanism for negative feedback regulation of PKC. We and for an of this We at Baylor for with the phosphopeptide