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The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent mannerCyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.
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Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrestCentrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).
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Cyclin-dependent kinase 5 phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95 in neuronsPSD-95 (postsynaptic density 95) is a postsynaptic scaffolding protein that links NMDA receptors to the cytoskeleton and signaling molecules. The N-terminal domain of PSD-95 is involved in the synaptic targeting and clustering of PSD-95 and in the clustering of NMDA receptors at synapses. The N-terminal domain of PSD-95 contains three consensus phosphorylation sites for cyclin-dependent kinase 5 (cdk5), a proline-directed serine-threonine kinase essential for brain development and implicated in synaptic plasticity, dopamine signaling, cocaine addiction, and neurodegenerative disorders. We report that PSD-95 is phosphorylated in the N-terminal domain by cdk5 in vitro and in vivo, and that this phosphorylation is not detectable in brain lysates of cdk5-/- mice. N-terminal phosphorylated PSD-95 is found in PSD fractions together with cdk5 and its activator, p35, suggesting a role for phosphorylated PSD-95 at synapses. In heterologous cells, coexpression of active cdk5 reduces the ability of PSD-95 to multimerize and to cluster neuronal ion channels, two functions attributed to the N-terminal domain of PSD-95. Consistent with these observations, the lack of cdk5 activity in cultured neurons results in larger clusters of PSD-95. In cdk5-/- cortical neurons, more prominent PSD-95 immunostained clusters are observed than in wild-type neurons. In hippocampal neurons, the expression of DNcdk5 (inactive form of cdk5) or of the triple alanine mutant (T19A, S25A, S35A) full-length PSD-95 results in increased PSD-95 cluster size. These results identify cdk5-dependent phosphorylation of the N-terminal domain of PSD-95 as a novel mechanism for regulating the clustering of PSD-95. Moreover, these observations support the possibility that cdk5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/NMDA receptors at synapses, thus providing a possible mechanism for rapid changes in density and/or number of receptor at synapses.
