Identification of the major multiphosphorylation site in mammalian neurofilaments.

Abstract

The sequence Lys-Ser-Pro-Val-Pro-Lys-SerPro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neuroframent (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs-i.e., MAP2 and tau.

Regional modulation of neurofilament organization by myelination in normal axons

“…All three ofthese proteins share a central rod domain, but they differ in their C-terminal regions. In NF-H, this region contains over 40 lysine-serine-proline repeats (Julien et al, 1986(Julien et al, , 1988Lees et al, 1988), which provide potential phosphorylation sites (Julien et al, 1983;Geisler et al, 1983;Lee et al, 1988). The extent of phosphorylation of this region, which contributes to the sidearms of the neurofilament (Hirokawa et al, 1984;Carden et al, 1985;Hisanaga and Hirokawa, 1988) appears to differ within different regions of the neuron, with a higher phosphorylation state in the axon than in the cell body and dendrite (Stemberger and Sternberger, 1983;Lee et al, 1986Lee et al, , 1987.…”

“…SMI-31 is directed against the extensively phosphorylated form of NF-H, the sequence motif containing highly phosphorylated lysine-serine-proline. The SMI-32 recognize a nonphosphorylated or relatively hypophosphorylated form of this motif on neurofilaments (Stemberaer and Stemberger, 1983;Lee et al, 1988).…”

Tau protein becomes long and stiff upon phosphorylation: correlation between paracrystalline structure and degree of phosphorylation.

“…Phosphorylation by a kinase which acts similarly to CaMK (in terms of reducing the electrophoretic mobility) acts on an epitope which tau shares with the paired helical filaments of Alzheimer's disease (Ishiguro et al, 1988), and the common region is in the COOH-terminal third of tau (Kondo et al, 1988). It is noteworthy that this part contains the sequence lys-ser-pro (residues 295-297), a sequence that is phosphorylated in neurofilaments and binds antibodies which cross-react between neurofilaments, MAP2, tau, and Alzheimer's tangles (Kosik et al, 1986;Lee et al, 1988b). Such an epitope occurs just downstream from the internal repeats of tan which form part of the potential microtubule-binding domain (compare Lee et al, 1988a;and Aizawa et al, 1988).…”

Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain

“…These include casein kinase I Hollander et al, 1996) and at least three different Pro-dependent kinases: protein kinase FA/glycogen synthase kinase-3 (GSK3; Guan et al, 1991), cyclin-dependent kinases (Hisanaga et al, 1991;Guan et al, 1992;Lew et a!., 1992;Shetty et al, 1993;Sun et a!., 1996), and mitogenactivated protein kinase (ERK kinase; Roder and Ingram, 1991;Chertoff et al, 1995). The phosphorylation sites are mostly in Ser residues in Lys-Ser-Pro (KSP) sequences (Geisler et a!., 1987;Lee et a!., 1988), which are repeated as many as 60 times in mammalian NF-H. Other, less frequently repeated, potential phosphorylation motifs have also been recognized, especially in NF-M, including KXSP and KXXSP (Shaw, 1991). It is not known if the different motifs, or different individual sites, have different specific functions.…”