Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.
References
[1]
Francis SH, Corbin JD (1994) Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol 56: 237-272. doi:10.1146/annurev.ph.56.030194.001321. PubMed: 8010741.
[2]
Scott JD (1991) Cyclic nucleotide-dependent protein kinases. Pharmacol Ther 50: 123-145. doi:10.1016/0163-7258(91)90075-W. PubMed: 1653962.
[3]
Brandon EP, Idzerda RL, McKnight GS (1997) PKA isoforms, neural pathways, and behaviour: making the connection. Curr Opin Neurobiol 7: 397-403. doi:10.1016/S0959-4388(97)80069-4. PubMed: 9232801.
[4]
Buxton IL, Brunton LL (1983) Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 258: 10233-10239. PubMed: 6309796.
[5]
Zhong H, Sia GM, Sato TR, Gray NW, Mao T et al. (2009) Subcellular dynamics of type II PKA in neurons. Neuron 62: 363-374. doi:10.1016/j.neuron.2009.03.013. PubMed: 19447092.
[6]
Wong W, Scott JD (2004) AKAP signalling complexes: focal points in space and time. Nat Rev Mol Cell Biol 5: 959-970. doi:10.1038/nrm1527. PubMed: 15573134.
[7]
Harada A, Teng J, Takei Y, Oguchi K, Hirokawa N (2002) MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction. J Cell Biol 158: 541-549. doi:10.1083/jcb.200110134. PubMed: 12163474.
[8]
Lebrand C, Dent EW, Strasser GA, Lanier LM, Krause M et al. (2004) Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron 42: 37-49. doi:10.1016/S0896-6273(04)00108-4. PubMed: 15066263.
[9]
Kao HT, Song HJ, Porton B, Ming GL, Hoh J et al. (2002) A protein kinase A-dependent molecular switch in synapsins regulates neurite outgrowth. Nat Neurosci 5: 431-437. PubMed: 11976703.
[10]
Shelly M, Cancedda L, Heilshorn S, Sumbre G, Poo MM (2007) LKB1/STRAD promotes axon initiation during neuronal polarization. Cell 129: 565-577. doi:10.1016/j.cell.2007.04.012. PubMed: 17482549.
[11]
Barnes AP, Lilley BN, Pan YA, Plummer LJ, Powell AW et al. (2007) LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons. Cell 129: 549-563. doi:10.1016/j.cell.2007.03.025. PubMed: 17482548.
[12]
Shelly M, Cancedda L, Lim BK, Popescu AT, Cheng PL et al. (2011) Semaphorin3A regulates neuronal polarization by suppressing axon formation and promoting dendrite growth. Neuron 71: 433-446. doi:10.1016/j.neuron.2011.06.041. PubMed: 21835341.
[13]
Chetkovich DM, Chen L, Stocker TJ, Nicoll RA, Bredt DS (2002) Phosphorylation of the postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 binding site of stargazin regulates binding to PSD-95 and synaptic targeting of AMPA receptors. J Neurosci 22: 5791-5796. PubMed: 12122038.
[14]
Choi J, Ko J, Park E, Lee JR, Yoon J et al. (2002) Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95. J Biol Chem 277: 12359-12363. doi:10.1074/jbc.M200528200. PubMed: 11805122.
[15]
Thomas MJ, Malenka RC (2003) Synaptic plasticity in the mesolimbic dopamine system. Philos Trans R Soc Lond B Biol Sci 358: 815-819. doi:10.1098/rstb.2002.1236. PubMed: 12740128.
[16]
Bradshaw NJ, Soares DC, Carlyle BC, Ogawa F, Davidson-Smith H et al. (2011) PKA phosphorylation of NDE1 is DISC1/PDE4 dependent and modulates its interaction with LIS1 and NDEL1. J Neurosci 31: 9043-9054. doi:10.1523/JNEUROSCI.5410-10.2011. PubMed: 21677187.
[17]
Bradshaw NJ, Ogawa F, Antolin-Fontes B, Chubb JE, Carlyle BC et al. (2008) DISC1, PDE4B, and NDE1 at the centrosome and synapse. Biochem Biophys Res Commun 377: 1091-1096. doi:10.1016/j.bbrc.2008.10.120. PubMed: 18983980.
[18]
Feng Y, Walsh CA (2004) Mitotic spindle regulation by Nde1 controls cerebral cortical size. Neuron 44: 279-293. doi:10.1016/j.neuron.2004.09.023. PubMed: 15473967.
[19]
Sasaki S, Mori D, Toyo-oka K, Chen A, Garrett-Beal L et al. (2005) Complete loss of Ndel1 results in neuronal migration defects and early embryonic lethality. Mol Cell Biol 25: 7812-7827. doi:10.1128/MCB.25.17.7812-7827.2005. PubMed: 16107726.
[20]
Pawlisz AS, Mutch C, Wynshaw-Boris A, Chenn A, Walsh CA et al. (2008) Lis1-Nde1-dependent neuronal fate control determines cerebral cortical size and lamination. Hum Mol Genet 17: 2441-2455. doi:10.1093/hmg/ddn144. PubMed: 18469343.
[21]
Stein JC, Farooq M, Norton WT, Rubin CS (1987) Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes. J Biol Chem 262: 3002-3006. PubMed: 3029098.
[22]
Letourneau PC, Shattuck TA (1989) Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones. Development 105: 505-519. PubMed: 2612362.
[23]
Ozer RS, Halpain S (2000) Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton. Mol Biol Cell 11: 3573-3587. doi:10.1091/mbc.11.10.3573. PubMed: 11029056.
[24]
Dehmelt L, Smart FM, Ozer RS, Halpain S (2003) The role of microtubule-associated protein 2c in the reorganization of microtubules and lamellipodia during neurite initiation. J Neurosci 23: 9479-9490. PubMed: 14573527.
[25]
Roger B, Al-Bassam J, Dehmelt L, Milligan RA, Halpain S (2004) MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain. Curr Biol 14: 363-371. doi:10.1016/j.cub.2004.01.058. PubMed: 15028210.
[26]
Tanaka Y, Kawahata K, Nakata T, Hirokawa N (1992) Chronological expression of microtubule-associated proteins (MAPs) in EC cell P19 after neuronal induction by retinoic acid. Brain Res 596: 269-278. doi:10.1016/0006-8993(92)91557-U. PubMed: 1467987.
[27]
Edson K, Weisshaar B, Matus A (1993) Actin depolymerisation induces process formation on MAP2-transfected non-neuronal cells. Development 117: 689-700. PubMed: 8392463.
[28]
Han J, Han L, Tiwari P, Wen Z, Zheng JQ (2007) Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP. J Cell Biol 176: 101-111. doi:10.1083/jcb.200607128. PubMed: 17200417.
[29]
Khuchua Z, Wozniak DF, Bardgett ME, Yue Z, McDonald M et al. (2003) Deletion of the N-terminus of murine map2 by gene targeting disrupts hippocampal ca1 neuron architecture and alters contextual memory. Neuroscience 119: 101-111. doi:10.1016/S0306-4522(03)00094-0. PubMed: 12763072.
[30]
Ren Y, Li R, Zheng Y, Busch H (1998) Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases. J Biol Chem 273: 34954-34960. doi:10.1074/jbc.273.52.34954. PubMed: 9857026.
[31]
Ryan XP, Alldritt J, Svenningsson P, Allen PB, Wu GY et al. (2005) The Rho-specific GEF Lfc interacts with neurabin and spinophilin to regulate dendritic spine morphology. Neuron 47: 85-100. doi:10.1016/j.neuron.2005.05.013. PubMed: 15996550.
[32]
Meiri D, Greeve MA, Brunet A, Finan D, Wells CD et al. (2009) Modulation of Rho guanine exchange factor Lfc activity by protein kinase A-mediated phosphorylation. Mol Cell Biol 29: 5963-5973. doi:10.1128/MCB.01268-08. PubMed: 19667072.
[33]
Luo L (2000) Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci 1: 173-180. doi:10.1038/35044547. PubMed: 11257905.
[34]
Lee HS, Komarova YA, Nadezhdina ES, Anjum R, Peloquin JG et al. (2010) Phosphorylation controls autoinhibition of cytoplasmic linker protein-170. Mol Biol Cell 21: 2661-2673. doi:10.1091/mbc.E09-12-1036. PubMed: 20519438.
[35]
Neukirchen D, Bradke F (2011) Cytoplasmic linker proteins regulate neuronal polarization through microtubule and growth cone dynamics. J Neurosci 31: 1528-1538. doi:10.1523/JNEUROSCI.3983-10.2011. PubMed: 21273437.
[36]
Chen WS, Yueh CY, Huang YA, Hwang E (2011) An inverted method for culturing dissociated mouse hippocampal neurons. Neurosci Res.
[37]
Malin SA, Davis BM, Molliver DC (2007) Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc 2: 152-160. doi:10.1038/nprot.2006.461. PubMed: 17401349.
[38]
Jones-Villeneuve EM, McBurney MW, Rogers KA, Kalnins VI (1982) Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells. J Cell Biol 94: 253-262. doi:10.1083/jcb.94.2.253. PubMed: 7107698.
