Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.T he atypical Cdc2-related protein kinase Cdk5 is ubiquitously expressed in mammalian cells and tissues (1). In noncycling cells, Cdk5 phosphorylates multiple substrates to control such diverse phenomena as cell signaling, adhesion and motility, cytoskeletal organization, protein trafficking, and membrane fusion and dynamic organization (2). The membrane-bound Cdk5 effectors, p35/p39, direct and activate the kinase to specific membrane targets via mechanisms that are not yet well understood (3-6). Cdk5 plays a crucial regulatory role in glucosestimulated insulin secretion in pancreatic cells (7,8). In addition, CDK5 is involved in the loss of ␤ cell function under glucotoxic conditions, revealing the potential therapeutic value of CDK5 inhibitors in the treatment of type 2 diabetes. Recently, fine mapping and genome-wide association studies have identified SNPs that affect a p35 homolog (9, 10) and calpain 10 (11) as being associated with type 2 diabetes susceptibility. The finding that the specific and strong Cdks inhibitor roscovitine inhibited 2DOG uptake by adipocytes has led us to investigate the involvement of Cdk5 in the regulation of glucose uptake in 3T3-L1 adipocytes.Here, we report that insulin stimulates Cdk5 activity in 3T3-L1 adipocytes, and knockdown of the kinase inhibits glucose uptake in these cells. Insulin-activated Cdk5 phosphorylates the synaptotagmin homolog E-Syt1 and promotes its association with GLUT4. Both E-Syt1 phosphorylation and GLUT4 association are inhibited by roscovitine. These findings are discussed in the context of the regulation of GLUT4-mediated glucose uptake in adipocytes, Cdk5 deregulation in response to calpains, and the susceptibility of families with the calpain 10 SNP-44 polymorphism to develop type 2 diabetes. ResultsCdk5 and p35 Are Coexpressed with GLUT4. We found comparable levels of Cdk5 transcript and protein in all GLUT4-expressing tissues and cells, including skeletal muscle, cardiac tissue, epidydimal white fat, and 3T3-L1 adipocytes (Fig. 1A). Both p35 transcript and protein were also detected in all samples (Fig. 1 A). The 32-kDa Cdk5 protein was abundant and unequally distributed among cytosolic (80%) plasma membrane (9%), microsomal (5%), and nuclear (6%) fractions [supporting information (SI) Fig. S1 A]. We identified 5 species of the Cdk5 activator p35, of which the 34-35...

The transition metal copper (Cu) is an essential trace element for all biota. Its redox properties bestow Cu with capabilities that are simultaneously essential and potentially damaging to the cell. Free Cu is virtually absent in the cell. The descriptions of the structural and functional organization of the metallothioneins, Cu-chaperones and P-type ATPases as well as of the mechanisms that regulate their distribution and functioning in the cell have enormously advanced our understanding of the Cu homeostasis and metabolism in the last decade. Cu is stored by metallothioneins and distributed by specialized chaperones to specific cell targets that make use of its redox properties. Transfer of Cu to newly synthesized cuproenzymes and Cu disposal is performed by the individual or concerted actions of the P-type ATPases ATP7A and ATP7B expressed in tissues. In mammalians liver is the major captor, distributor and excreter of Cu. Mutations in the P-type ATPases that interfere with their functioning and traffic are cause of the life-threatening Wilson (ATP7B) and Menkes (ATP7A) diseases.

Glucose entry into mammalian cells is facilitated by a family of glucose transport proteins known as GLUTs. Treatment of 3T3-L1 adipocytes with the Cdk5 inhibitor roscovitine strongly inhibits insulin-stimulated/GLUT4-mediated glucose transport. Inhibition of glucose uptake occurs within 2-6 min of the addition of roscovitine and is slowly reversed. The roscovitine treatment interferes with neither the translocation nor the insertion of GLUT4 into the plasma membrane. These studies support recent evidence showing that insulin-stimulated Cdk5 is implicated in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes.