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2.
Neurochem Res ; 47(9): 2668-2682, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35347634

RESUMO

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.


Assuntos
Axônios , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Regeneração Nervosa , Animais , Axônios/metabolismo , Proteína GAP-43/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mamíferos/metabolismo , Camundongos , Regeneração Nervosa/fisiologia , Fosforilação , Serina/metabolismo
3.
Mol Brain ; 14(1): 66, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33832520

RESUMO

GAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.


Assuntos
Axônios/metabolismo , Biomarcadores/metabolismo , Proteína GAP-43/metabolismo , Mamíferos/metabolismo , Fosfotreonina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Encéfalo/embriologia , Callithrix , Células Cultivadas , Furões , Proteína GAP-43/química , Cones de Crescimento/metabolismo , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos C57BL , Regeneração Nervosa , Fosforilação , Primatas , Nervo Isquiático/lesões
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