Cdk5 regulates the organization of Nestin and its association with p35.
The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.
Sahlgren
Cecilia M
CM
Department of Biology, Abo Akademi University, BioCity, Finland.
Mikhailov
Andrey
A
Vaittinen
Samuli
S
Pallari
Hanna-Mari
HM
Kalimo
Hannu
H
Pant
Harish C
HC
Eriksson
John E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology; D032446:Myoblasts / Q000166:cytology / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D064231:Nestin; D009474:Neurons / Q000166:cytology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012038:Regeneration / Q000502:physiology; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D014746:Vimentin; C089495:neuronal Cdk5 activator (p25-p35); D013912:Threonine; D051360:Cyclin-Dependent Kinase 5; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases