Characterization of Cdk2-cyclin E complexes in plasma membrane and endosomes of liver parenchyma. Insulin-dependent regulation.
Rat liver parenchyma Golgi/endosomes fractions harbor a tyrosine-phosphorylated 34-kDa protein. Screening of Golgi, endosomes (ENs), plasmalemma (PM), and cytosolic (Cyt) fractions revealed the presence of the mitotic kinase Cdk2 in ENs, PM, and Cyt. The fluid phase endocytic marker horseradish peroxidase gained access to the endosomal Cdk2, confirming its localization. Cdk2 was shown to be associated to cyclin E and was active in ENs and PM fractions. The administration of a single dose of insulin (1.5 microgram/100 g, body weight) induced a time-dependent activation of the insulin receptor kinase in these structures. Insulin receptor-kinase activation was followed by the inhibition of immunoprecipitated Cdk2-cyclin E kinase activity in PM and the progressive disappearance of cyclin E. In marked contrast, no such effect was observed in ENs. The injection of a phosphotyrosyl phosphatase inhibitor (bpV(phen)) increased the levels of cyclin E in ENs and PM. A massive recruitment of p27(kip1) was observed in the Cdk2-cyclin E complexes isolated from PM and Cyt but not from ENs. In vitro, Cdk2-cyclin E complexes have the capacity to inhibit the formation of hybrid structures containing horseradish peroxidase and radioiodinated epidermal growth factor. Therefore, in the PM and ENs of adult rat liver, an active and regulated pool of the mitotic kinase Cdk2-cyclin E and some yet to be defined effectors are present. Cdk2 may contribute to the modulation of transport events and/or maintenance of the topology of endocytic elements.
Gaulin
J F
JF
Unité de Recherche en Pédiatrie, Laboratoire de Biologie Cellulaire, Centre de Recherche du CHUL, Université Laval, Québec G1V 4G2, Canada.
Fiset
A
A
Fortier
S
S
Faure
R L
RL
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism*; D007328:Insulin / Q000502:physiology*; D008099:Liver / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D019927:Cyclin E; D007328:Insulin; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases