A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death

The intermediate filament protein, nestin, has been implicated as an organizer of survival-determining signaling molecules. When nestin expression was related to the sensitivity of neural progenitor cells to oxidant-induced apoptosis, nestin displayed a distinct cytoprotective effect. Oxidative stress in neuronal precursor cells led to downregulation of nestin with subsequent activation of cyclin-dependent kinase 5 (Cdk5), a crucial kinase in the nervous system. Nestin downregulation was a prerequisite for the Cdk5-dependent apoptosis, as overexpression of nestin efficiently inhibited induction of apoptosis, whereas depletion of nestin by RNA interference had a sensitizing effect. When the underlying link between nestin and Cdk5 was analyzed, we observed that nestin serves as a scaffold for Cdk5, with binding restricted to a specific region following the alpha-helical domain of nestin, and that the presence and organization of nestin regulated the sequestration and activity of Cdk5, as well as the ubiquitylation and turnover of its regulator, p35. Our data imply that nestin is a survival determinant whose action is based upon a novel mode of Cdk5 regulation, affecting the targeting, activity, and turnover of the Cdk5/p35 signaling complex

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

Introducing intermediate filaments: from discovery to disease

It took more than 100 years before it was established that the proteins that form intermediate filaments (IFs) comprise a unified protein family, the members of which are ubiquitous in virtually all differentiated cells and present both in the cytoplasm and in the nucleus. However, during the past 2 decades, knowledge regarding the functions of these structures has been expanding rapidly. Many disease-related roles of IFs have been revealed. In some cases, the molecular mechanisms underlying these diseases reflect disturbances in the functions traditionally assigned to IFs, i.e., maintenance of structural and mechanical integrity of cells and tissues. However, many disease conditions seem to link to the nonmechanical functions of IFs, many of which have been defined only in the past few years

Nestin as a regulator of Cdk5 in differentiating myoblasts

Our results show that the intermediate filament protein nestin regulates the early stages of myogenesis by a bidirectional interrelationship between nestin and Cdk5. Cdk5 regulates the organization and stability of its own scaffold nestin, which in turn controls the effects of Cdk5

Protein Kinase Cζ Regulates Cdk5/p25 Signaling during Myogenesis

Atypical protein kinase Cζ (PKCζ) is emerging as a mediator of differentiation. Here, we describe a critical role for PKCζ during myogenic differentiation. Our results identify PKCζ as a controller of myogenic differentiation by its regulation of Cdk5