Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases.
As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.
Veeranna
Center for Dementia Research, Nathan Kline Institute for Psychiatric Research, Orangeburg, New York, USA.
Lee
Ju-Hyun
JH
Pareek
Tej K
TK
Jaffee
Howard
H
Boland
Barry
B
Vinod
K Yaragudri
KY
Amin
Niranjana
N
Kulkarni
Ashok B
AB
Pant
Harish C
HC
Nixon
Ralph A
RA
D020816:Amino Acid Motifs / Q000276:immunology; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001921:Brain / Q000196:embryology* / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016900:Neurofilament Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D038461:Proline-Directed Protein Kinases / Q000276:immunology / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; C089495:neuronal Cdk5 activator (p25-p35); D038461:Proline-Directed Protein Kinases; D051360:Cyclin-Dependent Kinase 5; C495906:Cdk5 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D008239:Lysine