Membrane localization of 3-phosphoinositide-dependent protein kinase-1 stimulates activities of Akt and atypical protein kinase C but does not stimulate glucose transport and glycogen synthesis in 3T3-L1 adipocytes.
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.
Egawa
Katsuya
K
Third Department of Medicine and Department of Anatomy, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan.
Maegawa
Hiroshi
H
Shi
Kun
K
Nakamura
Takaaki
T
Obata
Toshiyuki
T
Yoshizaki
Takeshi
T
Morino
Katsutaro
K
Shimizu
Shinya
S
Nishio
Yoshihiko
Y
Suzuki
Eiji
E
Kashiwagi
Atsunori
A
D064413:3-Phosphoinositide-Dependent Protein Kinases; D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000502:physiology*; D001921:Brain / Q000502:physiology; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D006003:Glycogen / Q000096:biosynthesis*; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D051275:Glucose Transporter Type 4; D007328:Insulin; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; C060974:phosphatidylinositol 3,4,5-triphosphate; D006003:Glycogen; D064413:3-Phosphoinositide-Dependent Protein Kinases; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C115015:PKC-3 protein; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose