Characterization of Cdk2-Cyclin E Complexes in Plasma Membrane and Endosomes of Liver Parenchyma

Abstract

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 g/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.

Genome-Scale Identification of Membrane-Associated Human mRNAs

“…The protein encoded by ACK1 is localized to clathrin-containing vesicles [21], and the TXK protein binds lipid rafts via a palmitoylated cysteine-string motif [22]. Other examples included cyclin B3, whose family member cyclin B1 interacts with the cytoplasmic domain of the TM receptor PTCH [23], and cyclin E2, which has been shown to associate with endosomes and the plasma membrane of hepatocytes [24]. Thus, some genes, previously reported to be CN proteins and whose mRNAs were enriched in the membrane fraction, encode proteins that function in the cytosol, but in close association with membranes.…”

Activation of Stat3 by cell confluence reveals negative regulation of Stat3 by cdk2

“…Since cdk2 activity affects Stat3 phosphorylation, it most likely acts at a proximal point in the transduction pathway. In rat hepatocytes, Cdk2 has been reported to modulate receptor activation at the plasma membrane by regulating endocytosis (Gaulin et al, 2000). We have shown that the cdk2-inhibitor p27 also may interact with signaling complexes by localizing to lipid rafts (Yaroslavskiy et al, 2001) Such localization could facilitate 'inside-out' signaling of cell cycle regulators to alter signal transduction at the membrane.…”

MAPK Phosphorylation of Connexin 43 Promotes Binding of Cyclin E and Smooth Muscle Cell Proliferation

“…34 Accumulation of cyclinE-CDK2 complexes in s-phase of the cell cycle can be further regulated by MAPK activation. 35 In our studies we identified that PDGF temporally increases global expression of Cx43, Cx43-P, cyclin E and CDK2 proteins over a 24 hour period, and that Cx43 and cyclin E protein expression increased transiently in membrane fractions with marked reductions found between 12–24 hours.…”