Cyclin-dependent kinase 5 (cdk5) and its neuron-specific activator p35 are required for neurite outgrowth and cortical lamination. Proteolytic cleavage of p35 produces p25, which accumulates in the brains of patients with Alzheimer's disease. Conversion of p35 to p25 causes prolonged activation and mislocalization of cdk5. Consequently, the p25/cdk5 kinase hyperphosphorylates tau, disrupts the cytoskeleton and promotes the death (apoptosis) of primary neurons. Here we describe the mechanism of conversion of p35 to p25. In cultured primary cortical neurons, excitotoxins, hypoxic stress and calcium influx induce the production of p25. In fresh brain lysates, addition of calcium can stimulate cleavage of p35 to p25. Specific inhibitors of calpain, a calcium-dependent cysteine protease, effectively inhibit the calcium-induced cleavage of p35. In vitro, calpain directly cleaves p35 to release a fragment with relative molecular mass 25,000. The sequence of the calpain cleavage product corresponds precisely to that of p25. Application of the amyloid beta-peptide A beta(1-42) induces the conversion of p35 to p25 in primary cortical neurons. Furthermore, inhibition of cdk5 or calpain activity reduces cell death in A beta-treated cortical neurons. These observations indicate that cleavage of p35 to p25 by calpain may be involved in the pathogenesis of Alzheimer's disease.

The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.

The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an "inside-out" manner to produce six cortical laminae. We demonstrate here that p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase.