Abstract
It is reported that 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is activated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner and phosphorylates Akt, p70S6 kinase, and atypical protein kinase C (PKC), but its function on insulin signaling is still unclear. We cloned a full-length pdk-1 cDNA from a human brain cDNA library, and the adenovirus to overexpress wild type PDK-1 (PDK-1WT) or membrane-targeted PDK-1 (PDK-1CAAX) was constructed. Overexpressed PDK-1WT existed mainly at cytosol, and PDK-1CAAX was located at the plasma membrane. In 3T3-L1 adipocytes, insulin induced mobility shift of PDK-1 protein, but overexpressed PDK-1WT and CAAX were shifted at the basal state. Insulin stimulated tyrosine phosphorylation of PDK-1WT, but PDK-1CAAX was already tyrosine-phosphorylated at the basal state. Overexpression of PDK-1WT led to a full activation of PKC zeta/lambda without insulin stimulation but showed only the minimum effects to stimulate phosphorylation of Akt and GSK-3. In contrast, the overexpression of PDK-1CAAX caused phosphorylation of Akt and GSK-3 more strongly without insulin stimulation. However, PDK-1CAAX did not affect 2-deoxyglucose uptake and inhibited glycogen synthesis, surprisingly. Finally, PDK-1CAAX expression inhibited insulin-induced ERK1/2 phosphorylation in a dose-dependent manner. Taken together, the translocation of PDK-1 from cytosol to the plasma membrane is critical for Akt and GSK-3 activation. On the other hand, only atypical PKC and Akt activation was insufficient for stimulation of glucose transport, and constitutive activation of Akt-GSK-3 pathway may inhibit glycogen synthesis and MAPK cascade in 3T3-L1 adipocytes.
Highlights
One of the major effects of insulin is stimulation of glucose transport with translocation of glucose transporter 4 Glut4.1
It was reported that the overexpression of wild type PDK-1 by the electroporation method provoked the increases in the activity of cotransfected hemagglutinin-tagged protein kinase C (PKC) and concomitantly enhanced hemagglutinin-tagged Glut4 translocation in rat adipocytes [15]
Expression of PDK-1WT and PDK-1CAAX in Rat Primary Cultured Hepatocytes or 3T3-L1 Adipocytes—Isolated primary cultured hepatocytes were infected with recombinant adenovirus expressing wild type PDK-1, Ad5-PDK-1WT, or membranetargeted PDK-1, Ad5-PDK-1CAAX, at 10 m.o.i. for 1 h
Summary
Glucose transporter; PI, phosphatidylinositol; PDK-1, 3-phosphoinositide-dependent protein kinase-1; PKC, protein kinase C; ERK, extracellular signal-regulated. PDK-1 mediates signaling pathways between PI 3-kinase and its downstream molecules, and it is possible that PDK-1 plays an important role on insulin-stimulated glucose transport. Kinase; DME medium, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; GSK, glycogen synthase kinase-3; MBP, myelin basic protein; PDK-1WT, wild type PDK-1; PDK-1CAAX, membrane-targeted PDK-1; E1, ubiquitin-activating enzyme. Neither wild type nor membrane-targeted PDK-1 enhances glucose transport in 3T3-L1 adipocytes This finding suggests that PDK-1 stimulates atypical PKC and Akt by different mechanisms, and localization of PDK-1 is important for regulation of downstream signaling but another mechanism is still needed for total activation
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