Protein Kinase C Isoforms Are Translocated to Microtubules in Neurons

Activation of protein kinase C (PKC) increases microtubule (MT) growth lifetimes, resulting in extension of a nocodazole-sensitive population of MTs in Aplysia growth cones. We examined whether the two phorbol ester-activated PKCs inAplysia, the Ca2+-activated PKC Apl I and the Ca2+-independent PKC Apl II, are associated with these MTs. Phorbol esters translocated PKC to the Triton X-100-insoluble fraction, and a significant portion of this translocated pool was sensitive to low concentrations of nocodazole. Low doses of nocodazole had no effect on the amount of PKC in the Triton X-100-insoluble fraction in the absence of phorbol esters, whereas higher doses of nocodazole reduced basal levels of PKC Apl II. The F-actin cytoskeletal disrupter, latrunculin A, removed both PKCs from the Triton X-100-insoluble fraction in both control and phorbol ester-treated nervous systems. PKC Apl II also directly interacted with purified MTs. In detergent-extracted cells, both PKCs immunolocalized predominantly with MTs. PKCs were associated with newly formed MTs invading the actin-rich peripheral growth cone domain after PKC activation. Our results are consistent with a central role for PKCs in regulating MT extension. Activation of protein kinase C (PKC) increases microtubule (MT) growth lifetimes, resulting in extension of a nocodazole-sensitive population of MTs in Aplysia growth cones. We examined whether the two phorbol ester-activated PKCs inAplysia, the Ca2+-activated PKC Apl I and the Ca2+-independent PKC Apl II, are associated with these MTs. Phorbol esters translocated PKC to the Triton X-100-insoluble fraction, and a significant portion of this translocated pool was sensitive to low concentrations of nocodazole. Low doses of nocodazole had no effect on the amount of PKC in the Triton X-100-insoluble fraction in the absence of phorbol esters, whereas higher doses of nocodazole reduced basal levels of PKC Apl II. The F-actin cytoskeletal disrupter, latrunculin A, removed both PKCs from the Triton X-100-insoluble fraction in both control and phorbol ester-treated nervous systems. PKC Apl II also directly interacted with purified MTs. In detergent-extracted cells, both PKCs immunolocalized predominantly with MTs. PKCs were associated with newly formed MTs invading the actin-rich peripheral growth cone domain after PKC activation. Our results are consistent with a central role for PKCs in regulating MT extension. A large amount of research has been directed at how protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; MT, microtubule; P domain, peripheral domain; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); MES, 2-(N-morpholino)ethanesulfonic acid; PDBu, phorbol 12,13-dibutyrate; CSB, cytoskeletal stabilization buffer. 1The abbreviations used are: PKC, protein kinase C; MT, microtubule; P domain, peripheral domain; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); MES, 2-(N-morpholino)ethanesulfonic acid; PDBu, phorbol 12,13-dibutyrate; CSB, cytoskeletal stabilization buffer. regulates cytoskeletal plasticity (1Keenan C. Kelleher D. Cell. Signal. 1998; 10: 225-232Google Scholar). Most of this research has focused on the actin cytoskeleton because PKCs bind directly to actin filaments (2Blobe G.C. Stribling D.S. Fabbro D. Stabel S. Hannun Y.A. J. Biol. Chem. 1996; 271: 15823-15830Google Scholar, 3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar, 4Prekeris R. Mayhew M.W. Cooper J.B. Terrian D.M. J. Cell Biol. 1996; 132: 77-90Google Scholar) and a number of important actin regulatory proteins have been shown to be controlled by PKC phosphorylation (5Matsuoka Y., Li, X. Bennett V. J. Cell Biol. 1998; 142: 485-497Google Scholar, 6Allen L.A. Aderem A. EMBO J. 1995; 14: 1109-1120Google Scholar, 7Ng T. Parsons M. Hughes W.E. Monypenny J. Zicha D. Gautreau A. Arpin M. Gschmeissner S. Verveer P.J. Bastiaens P.I. Parker P.J. EMBO J. 2001; 20: 2723-2741Google Scholar). In contrast, roles for PKC in regulating microtubule (MT) function have not been extensively characterized. We recently reported an interesting and potentially important new role for PKC in regulating MT dynamics inAplysia growth cones (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). The growth cone is divided into three domains, a central domain rich in MTs, a transition zone, and a peripheral domain (P domain) rich in filamentous actin. Axonal MTs have a (+)-end distal orientation and undergo bouts of (+)-end assembly, which tend to drive them into the P domain against retrograde F-actin flow. The density of MTs in the P domain appears to be maintained at relatively low levels because they are continually transported rearward via coupling to retrograde F-actin flow (9Waterman-Storer C.M. Salmon E.D. J. Cell Biol. 1997; 139: 417-434Google Scholar, 10Forscher P. Smith S.J. J. Cell Biol. 1988; 107: 1505-1516Google Scholar). We recently reported that, after PKC activation, MTs tend to advance into the P domain because their average MT growth rates increase to exceed retrograde F-actin flow rates (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). The extension of MTs into the P domain may be important in axon extension, guidance, and/or delivery of important molecules to the plasma membrane (11Strong J.A. Fox A.P. Tsien R.W. Kaczmarek L.K. Nature. 1987; 325: 714-717Google Scholar, 12Knox R.J. Quattrocki E.A. Connor J.A. Kaczmarek L.K. Neuron. 1992; 8: 883-889Google Scholar, 13Rosner H. Fischer H. Neurosci. Lett. 1996; 219: 175-178Google Scholar). Examination of PKC action is simplified in the Aplysianervous system, as there are only two phorbol ester-activated PKCs, the Ca2+-activated PKC Apl I (homologous to PKCα, PKCβ, and PKCγ in vertebrates) and the Ca2+-independent PKC Apl II (homologous to PKCε and PKCη in vertebrates) (14Kruger K.E. Sossin W.S. Sacktor T.C. Bergold P.J. Beushausen S. Schwartz J.H. J. Neurosci. 1991; 11: 2303-2313Google Scholar, 15Sossin W.S. Diaz A.R. Schwartz J.H. J. Biol. Chem. 1993; 268: 5763-5768Google Scholar). Both isoforms of PKC co-assemble with actin in vitro, and this is enhanced by phorbol esters and by inhibitors of PKC phosphorylation (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). Using immunocytochemistry, PKC Apl II was localized to actin cables in the growth cone (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). Both PKCs also translocated to a Triton X-100-insoluble fraction by phorbol esters, and this was initially assumed to represent the actin cytoskeleton (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). However, given our recent report of PKC regulation of MT dynamics, we have further investigated the cytoskeletal pools with which PKC associates in neurons. We have also examined the distribution of PKC on MTs before and after PKC activation, using different cell extraction protocols that retain native associations of PKC with the cytoskeleton. We show that, upon activation by phorbol esters, both isoforms of PKC rapidly translocate onto newly assembled MTs, suggesting a direct role for PKC in regulating distal MT advance in neuronal growth cones. A. californica (50–200 g) were purchased from Marine Specimens Unlimited (Pacific Palisades, CA) or the Aplysia resource facility at the University of Miami and kept in an aquarium for at least 3 days before experimentation. The animals were first placed in a bath of isotonic MgCl2/artificial seawater (1:1, v/v) and then anesthetized by injection of isotonic MgCl2. The ganglia were dissected from the animals and trimmed of connective tissue in ice-cold dissecting medium (230 mm NaCl, 220 mmMgCl2, 1 mm CaCl2, 10 mm KCl, 10 mm HEPES, 0.2× low methionine amino acid mixture, 0.2× minimal essential medium nonessential amino acids (Invitrogen), 0.5× minimal essential medium vitamin solution, 0.1 mm glutamine, and 0.1% glucose, final pH 7.8). PKC was obtained for these experiments from the cytosol of Sf9 cells infected with baculovirus encoding PKC Apl I or Apl II as described (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). Purified bovine brain tubulin (10 mg/ml; ICN Biomedicals, Inc., Aurora, OH) was stored in 80 mm PIPES (sesquisodium salt), 1 mm EGTA, 1 mm MgCl2, 1 mm GTP, and 10% glycerol, pH 6.8, at −70 °C. Varying amounts of tubulin from the above stock solution were preincubated for 30 min at 37 °C with an equal volume of reassembly buffer (0.1 M MES, 1 mm EGTA, and 0.5 mm MgCl2) containing 1 mmGTP and 10% glycerol (RGG buffer) plus 10 μg/ml Taxol (Sigma) for some experiments. These samples were then further diluted in RGG buffer to obtain the desired concentration of microtubules. Finally, the samples were incubated in 1 μm 4β-phorbol 12,13-dibutyrate (PDBu; the PKC activator) or 1 μm4α-PDBu (the inactive analog of the phorbol ester) in RGG buffer with 10 μl of Sf9 cell cytosolic fraction expressing PKC Apl I or Apl II for 30 min at room temperature. The samples were centrifuged at 94,000 × g for 30 min at 25 °C. Supernatants were removed and added to 20 μl of sample buffer (2% SDS, 10% glycerol, 100 mm dithiothreitol, 60 mm Tris, pH 6.8, and 0.001% bromphenol blue). Following resuspension of the pellets, samples were loaded onto a 9% SDS-polyacrylamide gel. In Aplysia, most ganglia are symmetrically paired on either side of the animal. In all experiments, the ganglion on one side of the animal was used as a control for the corresponding one on the other side. The ganglia were desheathed to facilitate penetration of phorbol ester and transferred to resting medium (the same as dissecting medium, but with 460 mm NaCl, 11 mm CaCl2, and 55 mm MgCl2). The ganglia were incubated in either drug solution (300 nm nocodazole (Molecular Probes, Inc., Eugene, OR) and/or 5 μm latrunculin A (Molecular Probes, Inc.)) or vehicle in resting medium containing 10 mmglutamine and 0.1% glucose. Subsequently, phorbol esters were added to both samples to a final concentration of 1 μm 4β-PDBu to examine PKC translocation or 1 μm 4α-PDBu (inactive isomer) to examine basal PKC levels. Ganglia were incubated for 1 h at 15 °C; this leads to maximum translocation of PKC (16Sossin W.S. Schwartz J.H. Mol. Brain Res. 1994; 24: 210-218Google Scholar). The ganglia were then washed twice and homogenized in 120 μl of cold homogenization buffer (50 mm Tris-HCl (pH 10 mm MgCl2, 1 mm EGTA, 5 mm 20 μg/ml 5 and 0.1 mm The samples were centrifuged at °C for 3 min at × g to A sample (10 from was removed for of protein The were centrifuged at × 30 Supernatants were a sample was for of the protein and the 80 μl was added to 20 μl of sample buffer. were in 80 μl of Triton in homogenization buffer. The were incubated at °C for 15 min and then centrifuged at °C for 30 min at × Supernatants Triton were removed and added to 20 μl of sample buffer. The Triton were in 100 μl of sample buffer. protein amounts containing 10 of protein and and P containing the maximum amount to equal from control and samples were loaded onto 9% SDS-polyacrylamide and by and with to PKCs Apl I and Apl II were as described (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). The were then and with to actin Biomedicals, or tubulin were and was using the at We our with the of which the using the is the experiments that, after this were with to the amount of protein a of (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). the of protein in of the Triton and Triton was The in this with nocodazole and/or latrunculin A was then the paired were paired control and cell were and with as described (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). were as described (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar) with in resting medium, or cells were with Triton before as were washed with low resting medium mm NaCl, 10 mm KCl, 5 mmMgCl2, 15 mm HEPES, and 60 pH containing 5 mm for were then with Triton in cytoskeletal stabilization buffer containing 80 mm PIPES, 5 mm EGTA, 1 mm MgCl2, 10 μm 1 μm plus for 1 with CSB, cells were with in both were for MTs, and MTs and PKCs, a (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar) and (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar, 15Sossin W.S. Diaz A.R. Schwartz J.H. J. Biol. Chem. 1993; 268: 5763-5768Google Scholar) were used as described These on and PKCs in (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar, 15Sossin W.S. Diaz A.R. Schwartz J.H. J. Biol. Chem. 1993; 268: 5763-5768Google Scholar). was used for and and were used for MTs and PKCs, were from Probes, samples were with a and control no Phorbol esters translocate PKC to the Triton X-100-insoluble fraction of the nervous (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). Phorbol esters also increase the amount of tubulin that after ganglia are in that MTs (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). both the MTs in growth cones and the tubulin in the of phorbol esters are sensitive to low concentrations of nocodazole (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). These low concentrations of nocodazole MT dynamics, on MT 1998; Scholar, M.W. J. Neurosci. 1996; Scholar) or the amount of tubulin in the absence of phorbol esters (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). We examined whether low concentrations of nocodazole phorbol translocation of PKCs to the Triton X-100-insoluble ganglia were with either the or inactive phorbol ester in the or absence of had no effect on the amount of either PKC in the Triton X-100-insoluble fraction the inactive phorbol ester was used 1 in 1 In contrast, added in the of the phorbol nm nocodazole the amount of both PKCs Apl I and Apl II in the Triton X-100-insoluble fraction and the amount of both PKCs in the fraction 1 in In the of phorbol esters, nocodazole also the amount of tubulin in the Triton X-100-insoluble fraction, but nocodazole had no effect on the amount of tubulin in the Triton X-100-insoluble fraction in the absence of phorbol esters 1 in 1 These results that PKCs are translocated to a population of MTs by phorbol ester not the amount of PKC in the Triton either before or after phorbol ester 1 in 1 The phorbol translocation of PKCs to the Triton (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar, W.S. Schwartz J.H. Mol. Brain Res. 1994; 24: 210-218Google Scholar) is to either plasma membrane or and not be to be sensitive to of the cytoskeleton. We have shown that both Aplysia PKCs with actin filaments in a phorbol (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). further we examined the effect of latrunculin A, a that and actin filaments A. 219: on the amount of PKC translocated to the Triton X-100-insoluble ganglia were with either the or inactive control phorbol ester in the or absence of 5 μm latrunculin A. A the of PKCs Apl I and Apl II in the Triton X-100-insoluble fraction both in the and absence of phorbol esters in was a in the amount of actin in the Triton X-100-insoluble fraction in is to the amount of actin from the Triton X-100-insoluble fraction in other P. J. Biol. Chem. 1997; Scholar, A. J. Cell 1995; Scholar). A not a significant in the amount of PKC in the Triton membrane fraction either in the or absence of phorbol esters in These results that a significant fraction of the PKC that is Triton X-100-insoluble is sensitive to of the actin cytoskeleton. that the of nocodazole and latrunculin A were on different pools of PKC, we whether nocodazole or latrunculin A in the of the other ganglia were with phorbol esters plus nm nocodazole in the or absence of 5 μm latrunculin A with phorbol esters plus 5 μm latrunculin A in the or absence of nm nocodazole. We that the of the two cytoskeletal were of one Both the of PKCs in the Triton X-100-insoluble fraction in the of the other A and in 3 the to be a as the were nocodazole removed a higher of the Triton X-100-insoluble PKC, as the PKC was no because of the of latrunculin A to both control and there to be two pools of PKCs, one associated with actin filaments and one associated with microtubules. These experiments to the of PKC that is Triton X-100-insoluble and sensitive to cytoskeletal basal of PKC Apl II and of PKC Apl I were associated with the Triton X-100-insoluble of this kinase was sensitive to of the cytoskeleton in the of both nocodazole and there was PKC of PKC Apl II and 10 of PKC Apl I In the of phorbol esters, of PKC Apl II and of PKC Apl I were associated with the Triton X-100-insoluble fraction, amounts in the absence of phorbol increase in the of PKC associated with the Triton X-100-insoluble fraction is consistent with our results translocation of PKCs to the Triton X-100-insoluble fraction (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). A large amount of the PKC translocated to the Triton X-100-insoluble fraction after phorbol ester was sensitive to cytoskeletal inhibitors in the of both nocodazole and only of PKC Apl II and of PKC Apl I Triton These results that the cytoskeleton is a for PKC translocation inAplysia neurons. We whether the PKCs with purified MTs. PKCs were in Sf9 cells using baculovirus W.S. J. Neurosci. 1996; and the cytosol from Sf9 cells was incubated with purified was then and and of PKCs was PKC Apl II, but not PKC Apl significant with MTs and was not enhanced by phorbol esters The of PKC Apl II to tubulin that PKC Apl II be on MTs in the absence of phorbol we ganglia with concentrations of nocodazole for a in the absence of phorbol ganglia were with vehicle concentrations of nocodazole removed tubulin from the Triton X-100-insoluble fraction, consistent with of MTs in the cells also reduced the basal levels of PKC Apl II, but not of PKC Apl in the Triton X-100-insoluble fraction These results that PKC Apl II is associated with in the absence of phorbol of PKC Apl II to MTs in A, paired ganglia were with vehicle or μm nocodazole for h and as described in the to the of PKC Apl I or Apl II or tubulin in the Triton fraction was and used to the control and paired was a significant effect of nocodazole on PKC Apl II and tubulin but no for PKC Apl I obtain for we examined the of both PKCs in cell growth are were with 1 μm or 10 μm to or PKC with Triton and for PKC Apl II, MTs, and actin filaments not these MT advance and some of P domain F-actin were as reported (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google we were to a of either PKC Apl I not or Apl II with MTs these of PKC Apl II with F-actin in the P domain was and this to be in the of the PKC which to increase the levels of peripheral F-actin and Apl II A and C and These are in with our suggesting that the of PKC with F-actin is by kinase (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). The to in be to from a large pool of cytosolic or our to the we first cells with Triton in containing and Taxol to actin and MT after cells were washed to and for MTs, and both In cells with 1 μm PDBu, of both PKCs with MTs was the of PKC the growth cone resulting from of protein the cell extraction C and The protein may in to to M. J. S. A. Scholar). The PKC with MTs appears to be because PKC was associated with the F-actin cytoskeleton these also that MTs in the axon as as invading the distal P domain were associated with PKCs Apl I and Apl II. whether this kinase cells were with and for MTs, and PKC Apl I and Apl II to MTs in the of the PKC phorbol ester activation not to be a for PKC with MTs in the growth important from this is that and proteins are proteins for PKC in Aplysia neurons. our experiments were to growth these only a fraction of the used for and the cytoskeleton is an important for PKC neurons. PKCs bind directly to actin and to of the actin cytoskeleton (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar, S. Parker P.J. Scholar). The for PKC to MTs have not been only PKC Apl II interacted with purified MTs in vitro, the of PKCs Apl I and Apl II not to in growth cones. The of PKC Apl I from the Triton X-100-insoluble fraction with concentrations of nocodazole also that PKCs Apl I and Apl II to MTs with different PKCs have been associated with in other as T. H. M. T. 2001; Scholar, J. P. R. J. D. V. R. J. 2001; Scholar, M. J. J. Cell Res. 1997; Scholar). In was shown to with both in A. Kelleher D. J. 1998; Scholar) and in cells Parker P.J. Cell 1997; 8: Scholar). is important for cell in A. D. Kelleher D. 2001; Scholar). to our results with the Ca2+-activated PKC Apl of the Ca2+-activated with tubulin in cells were shown to be Parker P.J. J. Cell 1995; Scholar). We have shown that phorbol esters translocated both PKCs Apl I and Apl II to the Triton X-100-insoluble pool and that this was by low concentrations of nocodazole. for this is that PKC associates with MTs. Phorbol ester activation of PKC on MTs may in phosphorylation of a protein important in MT (+)-end (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). MT after phorbol ester to an increase in the number of for PKC and levels of new MT and of for PKC is consistent with the results in that both PKCs are associated with MTs their that there not to be PKC MT that into the P domain suggesting that PKC not bind to newly MTs (8Kabir N. Schaefer A.W. Nkhost A. Sossin W.S. Forscher P. J. Cell Biol. 2001; 152: 1033-1043Google Scholar). the increase in the of MTs the increase in PKCs in the Triton X-100-insoluble Our results with latrunculin A show that a fraction of PKC was associated with the actin cytoskeleton both before and after phorbol ester PKCs bind to purified actin in a phorbol (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). However, inhibitors of PKC of PKC with actin (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google suggesting that the translocation to actin may be and to in experiments. growth cones were and then and PKC Apl II to be with some concentration in in the P domain to increase after PKC consistent with our (3Nakhost A. Forscher P. Sossin W.S. J. Neurochem. 1998; 71: 1221-1231Google Scholar). However, these to MTs was not In contrast, we that cell extraction in PKC Apl I and Apl II with MTs, both after PKC activation and in the of PKC inhibitors These results that PKCs Apl I and Apl II with MTs and that this not on the of in the P domain were not because of relatively levels of cytosolic and PKC We the that the from of cytosolic that These may be by direct of dynamics in cells in that is important to that the of PKC and MTs in and is not MT as was in for PKC not MT in the growth cones also appears to be as there was no PKC to the F-actin in and These that of the PKC associated with F-actin in growth cones before extraction is and associated with the Triton cytoskeletal there is a pool of Triton actin in Aplysia growth cones A.W. N. Forscher P. J. Cell Biol. Scholar). In contrast, in the experiments Triton X-100-insoluble actin. pool of PKC to actin is either not in growth cones or sensitive in some other to the used for extraction and PKC activation by phorbol esters distal MT advance in the growth is that the of PKC with MTs is important for this In PKCs are associated with and MTs in the central are their and this is for T. 1997; Scholar, Cell Scholar, D. D. 2001; 20: Scholar). The large of PKCs associated with and actin filaments in that the cytoskeleton is one of the of PKC action in these In the be important to how the of PKC on is to neuronal delivery of and growth cone We for

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