Site‐Specific Phosphorylation of Neurofilament‐L Is Mediated by Calcium/Calmodulin‐Dependent Protein Kinase II in the Apical Dendrites During Long‐Term Potentiation

Abstract

Neurofilament-L (NF-L), one subunit of the neuronal intermediate filaments, is a major element of neuronal cytoskeletons. The dynamics of NF-L are regulated by phosphorylation of its head domain. The phosphorylation sites of the NF-L head domain by protein kinase A, protein kinase C, and Rho-associated kinase have been previously identified, and those by calcium/ calmodulin-dependent protein kinase II (CaMKII) were identified in this study. A series of site-and phosphorylation state-specific antibodies against NF-L was prepared to investigate NF-L phosphorylation in neuronal systems. Long-term potentiation (LTP) is a cellular model of neuronal plasticity that is thought to involve the phosphorylation of various proteins. NF-L is considered a possible substrate for phosphorylation. During LTP stimulation of mouse hippocampal slices, the series of antibodies demonstrated the increase in the phosphorylation level of Ser 57 in NF-L and the visualization of the localized distribution of Ser 57 phosphorylation in a subpopulation of apical dendrites of the pyramidal neurons. Furthermore, Ser 57 phosphorylation during LTP is suggested to be mediated by CaMKII. Here we show that NF-L is phosphorylated by CaMKII in a subpopulation of apical dendrites during LTP, indicating that Ser 57 is a novel phosphorylation site of NF-L in vivo related to the neuronal signal transduction. Key Words: Neurofilament-LPhosphorylation-Site-and phosphorylation state-specific antibody-Calcium/calmodulin-dependent protein kinase II-Long-term potentiation-Apical dendriteIntermediate filament. J. Neurochem. 75, 373-382 (2000).Intermediate filaments (IFs) constitute one of the three major cytoskeletal elements. Several protein kinases have been described to act as IF kinases in vivo, and the site-specific phosphorylation of IF proteins is thought to be responsible for cytoskeletal reorganization (Inagaki et al., 1987). During mitosis, a marked change in the phosphorylation state of IF proteins was found to accompany the structural changes of cells (Chou et al., 1991;Eriksson et al., 1992). A minor change in the phosphorylation state of IF proteins is assumed to play an important role in the interaction of IF proteins with IF-associated proteins and other proteins in organelles and the cytoskeleton (Hisanaga and Hirokawa, 1990;Gyoeva and Gelfand, 1991). A series of site-and phosphorylation state-specific antibodies, which recognize a phosphorylated residue and its flanking sequence, is a powerful tool for visualizing site-specific IF phosphorylation in cells and for identifying IF kinases in vivo (Nishizawa et al., 1991;Liao et al., 1995;Goto et al., 1998). Neurofilament, a neuronal IF, is abundantly distributed throughout the axon, perikaryon, and dendrite (Fuchs and Weber, 1994;Lee and Cleveland, 1996). Dynamic equilibrium and phosphorylation of the head domain of neurofilament-L (NF-L) regulate the organization of NF-L filaments, the core-forming components (Hisanaga and Hirokawa, 1988;Angelides et al., 1989;Liem, 1990;Nixon and Siha...

Neurofilaments at a glance

“…The head domains of NFL, NFM, NFH and a-internexin are phosphorylated and glycosylated (Dong et al, 1996;Manser et al, 2008;Tanaka et al, 1993;Vosseller et al, 2006). Phosphorylation of the NF head domain is mediated by the second messenger dependent kinases protein kinase A (PKA) and C (PKC) (Sihag et al, 1988;Sihag and Nixon, 1989;Sihag and Nixon, 1990) and possibly also by Cam kinase II (Hashimoto et al, 2000). The occurrence of NF head phosphorylation in the cell body soon after subunit synthesis reflects its suggested role in maintaining the disassembled state of NFs, as head phosphorylation of NFL inhibits NF assembly (Hashimoto et al, 2000;Hisanaga et al, 1990;Sihag et al, 1999;Sihag and Nixon, 1991).…”

“…Phosphorylation of the NF head domain is mediated by the second messenger dependent kinases protein kinase A (PKA) and C (PKC) (Sihag et al, 1988;Sihag and Nixon, 1989;Sihag and Nixon, 1990) and possibly also by Cam kinase II (Hashimoto et al, 2000). The occurrence of NF head phosphorylation in the cell body soon after subunit synthesis reflects its suggested role in maintaining the disassembled state of NFs, as head phosphorylation of NFL inhibits NF assembly (Hashimoto et al, 2000;Hisanaga et al, 1990;Sihag et al, 1999;Sihag and Nixon, 1991). Phosphorylation of the NF head domain is also known to modulate their interaction with fodrin, an important protein of the sub-axolemmal cytoskeleton (Frappier et al, 1987).…”

Differential expression and localization of the phosphorylated and nonphosphorylated neurofilaments during the early postnatal development of rat hippocampus

“…NF proteins, in particular NF-M and NF-H, are phosphorylated extensively, with highly conserved phosphorylation sites (Julien and Mushynski, 1983;Carden et al, 1985;Schlaepfer, 1987). It is suggested that the phosphorylation plays an important role in stabilizing and continuously remodeling NF filaments during the axonal growth and might be connected to plasticity of both axons and dendrites (Schlaepfer, 1987;Wang et al, 1994;Hashimoto et al, 2000;Posmantur et al, 2000).…”

In Vivo and in Vitro Phosphorylation at Ser-493 in the Glutamate (E)-segment of Neurofilament-H Subunit by Glycogen Synthase Kinase 3β

“…Ser-501 is in the SP sequence followed by Lys at the third C-terminal site. The SPXK sequence is a consensus phosphorylation motif for Cdk5 (17,18,20) and also mitogenactivated protein kinase. Because Ala substitution of Ser-501 reduced the phosphorylation of the E-segment by Cdk5 and ERK (data not shown), we suggest that this site would be phosphorylated by Cdk5 and ERK.…”

“…The phosphorylation of the head domain by second messenger-dependent protein kinases induces filament disassembly in vitro (13)(14)(15). Nascent NF-L is phosphorylated at Ser-55 by PKA until incorporation into the axonal NF network (16) and at Ser-57 by calcium/calmodulin-dependent protein kinase II (CaMKII) at apical dendrites of the pyramidal neurons in a hippocampal slice culture when long term potentiation is induced (17). The long C-terminal tail domains of two larger subunits, NF-M and NF-H, are highly phosphorylated in axons.…”