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.
Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals. We identify here, by MS, several tyrosine‐phosphorylated proteins in a rat liver Golgi/endosome (G/E) fraction including DPP IV. We show that a pool of DPP IV is tyrosine‐phosphorylated. Maximal phosphorylation was observed after 2 min following intravenous insulin injection. DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c‐Src) with maximal association also observed after 2 min following insulin injection. DPP IV was found phosphorylated after incubation of nonsolubilized G/E membranes with [γ‐32P]ATP. The c‐Src inhibitor PP2 inhibited DPP IV phosphorylation. Oriented proteolysis experiments indicate that a large pool of c‐Src is protected in G/E fractions. Following injection of the protein‐tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G/E fractions. In the fraction designated Lh, DPP IV levels decreased by 50% 15 min following insulin injection. Therefore, a pool of DPP IV is tyrosine‐phosphorylated in an insulin‐dependent manner. The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c‐Src‐containing signalling platforms.
A role for Src Family Kinases (SFKs) in the dynamics of endocytic and secretory pathways has previously been reported. Identification of low-abundance compartmentalized complexes still remains challenging, highlighting the need for novel tools. Here we describe analysis of SFK-signaling complexes of hepatic Golgi/endosomes (G/E) fractions by sequential affinity enrichment of proteins. Mouse G/E permeabilized membranes were first validated in terms of electron microscopy, 1-D electrophoresis (1-DE), insulin-mediated endocytosis and protein content. With the use of quantitative N-terminal labeling of tryptic peptides (iTRAQ), 1-DE and IEF tryptic peptides separation methods, a total of 666 proteins were identified, including the SFK Lyn. Following insulin injection, a series of proteins were recognized by an anti-phosphotyrosine antibody (alpha P42-2) raised against the residue most frequently phosphorylated by SFK on the adenoviral protein E4orf4 and that cross-reacts with endosomal SFK targets. By using affinity chromatography coupled with mass spectrometry, we identified 16 proteins classified as (1) recycling receptors, (2) vesicular trafficking proteins, (3) actin network proteins, (4) metabolism proteins, or (5) signaling proteins. One of these proteins, low density lipoprotein-related protein 1 (LRP1), which is a known SFK substrate, was found to associate with the internalized insulin receptor (IR), suggesting the presence of a co-internalization process. The identification of these proteomes should, thus, contribute to a better understanding of the molecular mechanisms that regulate trafficking events and insulin clearance.
During interphase the transport of material between different intracellular organelles requires accurate regulation of fusiogenic domains. Recent studies on hepatic endosomes indicated that compartmentalized Cdk2-cyclin E complexes act by braking fusion events. These Cdk2 complexes integrate tyrosine phosphorylation and dephosphorylation inputs, resulting in the control of the number of rounds of fusion at discrete domains. This leads to changes in the intracellular location of internalized receptors and ultimately their biological response.
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