p85α Gene Generates Three Isoforms of Regulatory Subunit for Phosphatidylinositol 3-Kinase (PI 3-Kinase), p50α, p55α, and p85α, with Different PI 3-Kinase Activity Elevating Responses to Insulin

doi:10.1074/jbc.272.12.7873

p85α Gene Generates Three Isoforms of Regulatory Subunit for Phosphatidylinositol 3-Kinase (PI 3-Kinase), p50α, p55α, and p85α, with Different PI 3-Kinase Activity Elevating Responses to Insulin*

From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, The
^‡ Institute for Adult Disease, Asahi Life Foundation, 1-9-14, Nishishinjuku, Shinjuku-ku, Tokyo 160, the
^§ Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gumma University, Showa-machi, Maebashi, Gumma 371, the
^¶ Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144, Kogushi, Ube, Yamaguchi 755, Japan

^∥To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3133); Fax: 81-3-5803-1874.

Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using ³²P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85α, but the SH3 and bcr homology domains of p85α were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85α gene product. We suggest that this protein be called p50α. Northern blotting using a specific DNA probe corresponding to p50α revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50α protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50α. Taken together, these observations indicate that the p85α gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85α, p55α, and p50α), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50α forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85α and p55α associate only with p110 that can be wortmannin-labeled. Furthermore, p50α exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50α expression in the liver and its marked responsiveness to insulin, p50α appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three α isoforms has a different function and may have specific roles in various tissues.

Footnotes

↵* This work was supported by Grant-in-Aid 08671136 for Scientific Research (to T. A.) from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) D78486[GenBank].
↵¹ The abbreviations used are:

PI 3-kinase

phosphatidylinositol 3-kinase

IRS-1

insulin receptor substrate-1

kb

kilobase pair(s)

PAGE

polyacrylamide gel electrophoresis

PMSF

phenylmethylsulfonyl fluoride

HA

hemagglutinin

IR

insulin receptors

CHO

Chinese hamster ovary

DIG

digoxigenin

N-SH2

N-terminal SH2.
- Received November 27, 1996.