Binding of brain spectrin to the 70‐kDa neurofilament subunit protein

Abstract

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in v i m assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 1251-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin.a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/ 50 -t 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x lo-' M.b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8 -7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Caz+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2 Brain spectrin of fodrin is a major protein in neurons, where it represents 3% of the membrane protein [l]. By immunofluorescence microscopy, fodrin has been localized and found associated to the plasmalemma in cell bodies, dendrites, axons and nerve terminals [2, 31. This association appears to involve two membrane-attachment proteins, ankyrin and synapsin I. They have been recently purified from brain [4, 51 and shown to possess the same functional properties as those described for ankyrin and protein 4.1 in erythrocytes: their presence suggests that, like erythrocyte spectrin, fodrin may play an active role in stabilizing the membrane of neurons as well as in immobilizing some integral membrane proteins as receptors, which are yet to be characterized.Unlike erythrocyte spectrin, fodrin is mobile and undergoes axonal transport. Axonal transport studies [6] have shown that fodrin is the only known protein to migrate at different rates along the axon, with vesicles, organelles and mitochondria (50 mm/day), with actin and myosin (5 mm/ day) and with microtubules and neurofilaments (0.5 -I mm/ Correspondence to L. A. Pradel, Institut de Biologie PhysicoChimique, 13 Rue Pierre el Marie Curie, F-75005 Paris, FranceAbbreviations. NF-L, NF-M, NF-H, 70-kDa, 160-kDa, 200-kDa neurofilament subunits; GFP, glial filament protein; BNPS-skatole, 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; PMSF, phenylmethylsulphonylfluoride. day). These various rates of migration can be explained if fodrin, a multifunctional protein, is capable of...

Neurofilament Transport Is Dependent on Actin and Myosin

“…Recent observations identify NFs as a major ligand of myosin in situ (Rao et al, 2002). NFs also have been shown to bind the actin-associated protein spectrin (Frappier et al, 1987(Frappier et al, , 1991. These observations, coupled with the observation that some NFs and punctate structures containing NF subunits translocate along the peripheral area of axonal neurites (Yabe et al, 2001a,b), leave open the possibility that the submembrane actin cortex and/or its associated motor myosin may facilitate axonal transport of NFs.…”

Functional diversity among spectrin isoforms

“…Erythroid and nonerythroid spectrins have been shown to interact directly with three classes of intermediate filaments (IF): desmin [Langley and Cohen, 19861, vimentin [Langley and Cohen, 19871, and neurofilaments [Frappier et al, 1987;Henmann and Wiche, 1987;Langley and Cohen, 19871. The molecular basis of these spectrin-IF associations remains unclear.…”

Dynamics of neuronal intermediate filaments: A developmental perspective

“…Certain of these sites completely turn over their phosphates in vivo without hours after synthesis, before neurofilaments have moved a considerable distance into the axon [Sihag and Nixon, 1989, 19911. It seems likely that some head domain sites are binding sites for other proteins, although none has yet been reported, and, remarkably, most of the binding sites for cytoskeletal proteins have so far been found on NF-L [Frappier et al, 1987[Frappier et al, , 1991Heimann et al, 1985;Miyata et al, 19861. Another possible function of NF-M head domain sites regulated by second messenger-dependent kinases may be to stabilize the assembled state of the neurofilament and to reduce its sensitivity to phosphorylation-induced disassembly [Nixon and Sihag, 19911-a property that may be disadvantageous to the long-lived filaments that support the axon.…”

“…This process would be expected to hinder neurofilament movement sterically , possibly to reduce attachment to the transport vector, and to increase accessibility to new binding sites for cross-linking proteins all along the filament [Nixon and Sihag, 19911. Supporting this hypothesis are studies that neurofilament transport rates vary in the predicted ways when the phosphorylation state of the C-terminal domain of NF-H changes during axonal maturation and in neurotoxic states [Watson et al, 1989, 19911. Presumably, stationary neurofilaments are interconnected with microtubules and other structures via microtubule-associated proteins and membrane cytoskeleta1 proteins [Nixon, 1987;Nixon et al, 19901, since binding sites for these are present on neurofilament subunits [Frappier et al, 1987;Miyata et al, 1986;Heimann et al, 19851. The existence of multiple phosphorylated isoforms of NF-M and NF-H, some of which are preferentially associated with transported neurofilaments and others with stationary neurofilaments, raises the possibility that neurofilament networks may be capable of undergoing dynamic rearrangement by altering the strength and varieties of interactions with other cytoskeleton proteins by means of phosphateinduced charge alterations and conformational changes that expose particular binding sites [Eyer and Leterrier, 1988;Hisanaga and Hirokawa, 1989, 19901.…”