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Molecular and Cellular Biology, December 1998, p. 6971-6982, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Requirement of Atypical Protein Kinase Clambda for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Ko Kotani,1 Wataru Ogawa,1,* Michihiro Matsumoto,1 Tadahiro Kitamura,1 Hiroshi Sakaue,1 Yasuhisa Hino,1 Kazuaki Miyake,1 Wataru Sano,1 Kazunori Akimoto,2 Shigeo Ohno,2 and Masato Kasuga1

Second Department of Internal Medicine, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017,1 and Department of Molecular Biology, Yokohama City University School of Medicine, Kanazawa-ku, Yokohama 236-0004,2 Japan

Received 25 February 1998/Returned for modification 15 April 1998/Accepted 14 August 1998

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambda KD or lambda Delta NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda , indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambda KD or lambda Delta NKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was ~50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambda Delta PD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambda Delta PD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda . These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

* Corresponding author. Mailing address: Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone: 81-78-341-7451. Fax: 81-78-382-2080. E-mail: ogawa{at}med.kobe-u.ac.jp.

Molecular and Cellular Biology, December 1998, p. 6971-6982, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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