BADERC-Lewis Cantley

Lewis Cantley, Ph.D.

Role of Phosphoinositide 3-Kinase in Cellular Regulation

The long term goal of research in this laboratory has been to understand the role of the phosphoinositide 3-kinase (PI3K) signaling pathway in cellular responses to growth factors. We discovered PI3K more than 15 years ago because of its co-purification with activated protein-tyrosine kinases. Work from this laboratory and other laboratories over the past 5 to 10 years has firmly established the importance of the PI3K pathway in cancers and in insulin signaling. Advances have also been made in elucidating a branching network of signals downstream of PI3K. Our recent studies of mice in which PI3K genes have been deleted have revealed the importance of PI3K in B cell development, stimulation and survival and have uncovered an unexpected role for the regulatory subunit of PI3K in suppressing insulin signaling. Although previous studies of cells in culture have indicated that PI3K is critical for most insulin responses, including GLUT4 recruitment to the cell membrane and glucose uptake, we found that insulin sensitivity was increased rather than decreased in mice lacking all isoforms of p85a. The increased insulin sensitivity was observed in insulin tolerance tests and in glucose tolerance tests. In addition, the p85a/p55a/p50a-/- mice had low insulin and low glucose after overnight fasting compared to wild type littermates. To further investigate the effect of loss of p85a isoforms on insulin sensitivity, we crossed the p85a/p55a/p50a-/- mice with mice heterozygous for insulin receptor (IR+/-) and heterozygous for IRS-1 (IRS-1+/-). A majority of the IR+/-, IRS-1+/- mice develop diabetes within 5 months of age as judged by an elevation in serum glucose. However, very few of the triple het mice (IR+/-, IRS-1+/-, p85a/p55a/p50a+/-) showed elevated serum glucose (Mauvais-Jarvis et al., 2002). Thus, even a partial loss of p85a isoforms improves insulin signaling enough to prevent diabetes in a mouse model. A possible explanation for these results is that the remaining p85b in the p85a/p55a/p50a-/- mice is more efficient than 85a in mediating insulin responses. Consistent with this idea, p85b was somewhat elevated in the p85a/p55a/p50a-/- mice, although not enough to compensate for the loss of p85a isoforms (Fruman et al., 2000). To test this model, we generated mice in which the p85b gene was deleted (p85b-/- mice). The phenotype of the p85b-/- mice is inconsistent with the model that p85b is more important than p85a in mediating insulin responses. The p85b-/- mice are viable, capable of reproducing and of a normal life span. However, they are approximately 10% smaller than wild type littermates. The p85b-/- mice had a normal glucose tolerance test but, like the p85a/p55a/p50a-/- mice showed increased insulin sensitivity in insulin tolerance tests (Ueki et al., 2002). Consistent with p85b being much less abundant than p85a isoforms in liver and muscle, we found very little change in total class Ia PI3K or in anti-phosphoTyr or anti-IRS-1 precipitable PI3K activity in the muscle or liver of insulin-injected p85b-/- mice compared with wild type littermates (Ueki et al., 2002). Curiously, we found a prolonged tyrosine phosphorylation of IRS-2 and prolonged association of PI3K activity with IRS-2 in muscle from insulin injected p85-/- mice compared to wild type littermates. Consistent with this observation, we also observed a small increase in insulin-dependent AKT activation in the muscle of p85b-/- mice compared to wild type littermates (Ueki et al., 2002).Taken together, these studies all indicate that reduction of any isoform of p85 results in an increase in insulin sensitivity. This observation raises the possibility that in addition to its role in recruiting p110 catalytic subunits of PI3K and regulating their activity, the p85 subunit may be playing a negative role in insulin signaling.A critical question raised by these studies is whether the increased insulin sensitivity observed in p85-/- mice is a tissue autonomous effect. To address this question, we have generated mice in which the p85a gene is floxed and can be deleted in a tissue-specific manner. We have crossed these mice with mice expressing Cre recombinase in skeletal muscle and with mice expressing Cre recombinase in fat. We will characterize insulin responses in these mice. Since skeletal muscle is responsible for most glucose clearance in mice, muscle-specific loss of p85a should result in increased insulin sensitivity if the effect is tissue autonomous. An alternative possibility is that loss of p85a in fat causes release of a factor that alters the insulin sensitivity in skeletal muscle. These studies should resolve this question and suggest new directions of research.

References:

1. Fruman DA, Snapper SB, Yballe CM, Davidson L, Yu JY, Alt FW, Cantley LC. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85alpha. Science. 1999 Jan 15;283(5400):393-7.

2. Mauvais-Jarvis F, Ueki K, Fruman DA, Hirshman MF, Sakamoto K, Goodyear LJ, Iannacone M, Accili D, Cantley LC, Kahn CR. Reduced expression of the murine p85alpha subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. J Clin Invest. 2002 Jan;109(1):141-9.

3. Ueki K, Fruman DA, Brachmann SM, Tseng YH, Cantley LC, Kahn CR. Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival. Mol Cell Biol. 2002 Feb;22(3):965-77.

4. Fruman DA, Mauvais-Jarvis F, Pollard DA, Yballe CM, Brazil D, Bronson RT, Kahn CR, Cantley LC. Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha. Nat Genet. 2000 Nov;26(3):379-82.

5. Hallmann D, Trumper K, Trusheim H, Ueki K, Kahn CR, Cantley LC, Fruman DA, Horsch D. Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha. J Biol Chem. 2003 Feb 14;278(7):5099-108. Epub 2002 Nov 14.

6. Ueki K, Yballe CM, Brachmann SM, Vicent D, Watt JM, Kahn CR, Cantley LC. Increased insulin sensitivity in mice lacking p85beta subunit of phosphoinositide 3-kinase. Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):419-24.

7. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 2002 Jul;10(1):151-62.

8. Chang JD, Field SJ, Rameh LE, Carpenter CL, Cantley LC. Identification and characterization of a phosphoinositide phosphate kinase homolog. J Biol Chem. 2004;279:11672-9.

9. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley LC. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A. 2004;101:3329-35.






			Lewis Cantley, Ph.D. Role of Phosphoinositide 3-Kinase in Cellular Regulation The long term goal of research in this laboratory has been to understand the role of the phosphoinositide 3-kinase (PI3K) signaling pathway in cellular responses to growth factors. We discovered PI3K more than 15 years ago because of its co-purification with activated protein-tyrosine kinases. Work from this laboratory and other laboratories over the past 5 to 10 years has firmly established the importance of the PI3K pathway in cancers and in insulin signaling. Advances have also been made in elucidating a branching network of signals downstream of PI3K. Our recent studies of mice in which PI3K genes have been deleted have revealed the importance of PI3K in B cell development, stimulation and survival and have uncovered an unexpected role for the regulatory subunit of PI3K in suppressing insulin signaling. Although previous studies of cells in culture have indicated that PI3K is critical for most insulin responses, including GLUT4 recruitment to the cell membrane and glucose uptake, we found that insulin sensitivity was increased rather than decreased in mice lacking all isoforms of p85a. The increased insulin sensitivity was observed in insulin tolerance tests and in glucose tolerance tests. In addition, the p85a/p55a/p50a-/- mice had low insulin and low glucose after overnight fasting compared to wild type littermates. To further investigate the effect of loss of p85a isoforms on insulin sensitivity, we crossed the p85a/p55a/p50a-/- mice with mice heterozygous for insulin receptor (IR+/-) and heterozygous for IRS-1 (IRS-1+/-). A majority of the IR+/-, IRS-1+/- mice develop diabetes within 5 months of age as judged by an elevation in serum glucose. However, very few of the triple het mice (IR+/-, IRS-1+/-, p85a/p55a/p50a+/-) showed elevated serum glucose (Mauvais-Jarvis et al., 2002). Thus, even a partial loss of p85a isoforms improves insulin signaling enough to prevent diabetes in a mouse model. A possible explanation for these results is that the remaining p85b in the p85a/p55a/p50a-/- mice is more efficient than 85a in mediating insulin responses. Consistent with this idea, p85b was somewhat elevated in the p85a/p55a/p50a-/- mice, although not enough to compensate for the loss of p85a isoforms (Fruman et al., 2000). To test this model, we generated mice in which the p85b gene was deleted (p85b-/- mice). The phenotype of the p85b-/- mice is inconsistent with the model that p85b is more important than p85a in mediating insulin responses. The p85b-/- mice are viable, capable of reproducing and of a normal life span. However, they are approximately 10% smaller than wild type littermates. The p85b-/- mice had a normal glucose tolerance test but, like the p85a/p55a/p50a-/- mice showed increased insulin sensitivity in insulin tolerance tests (Ueki et al., 2002). Consistent with p85b being much less abundant than p85a isoforms in liver and muscle, we found very little change in total class Ia PI3K or in anti-phosphoTyr or anti-IRS-1 precipitable PI3K activity in the muscle or liver of insulin-injected p85b-/- mice compared with wild type littermates (Ueki et al., 2002). Curiously, we found a prolonged tyrosine phosphorylation of IRS-2 and prolonged association of PI3K activity with IRS-2 in muscle from insulin injected p85-/- mice compared to wild type littermates. Consistent with this observation, we also observed a small increase in insulin-dependent AKT activation in the muscle of p85b-/- mice compared to wild type littermates (Ueki et al., 2002).Taken together, these studies all indicate that reduction of any isoform of p85 results in an increase in insulin sensitivity. This observation raises the possibility that in addition to its role in recruiting p110 catalytic subunits of PI3K and regulating their activity, the p85 subunit may be playing a negative role in insulin signaling.A critical question raised by these studies is whether the increased insulin sensitivity observed in p85-/- mice is a tissue autonomous effect. To address this question, we have generated mice in which the p85a gene is floxed and can be deleted in a tissue-specific manner. We have crossed these mice with mice expressing Cre recombinase in skeletal muscle and with mice expressing Cre recombinase in fat. We will characterize insulin responses in these mice. Since skeletal muscle is responsible for most glucose clearance in mice, muscle-specific loss of p85a should result in increased insulin sensitivity if the effect is tissue autonomous. An alternative possibility is that loss of p85a in fat causes release of a factor that alters the insulin sensitivity in skeletal muscle. These studies should resolve this question and suggest new directions of research. References: 1. Fruman DA, Snapper SB, Yballe CM, Davidson L, Yu JY, Alt FW, Cantley LC. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85alpha. Science. 1999 Jan 15;283(5400):393-7. 2. Mauvais-Jarvis F, Ueki K, Fruman DA, Hirshman MF, Sakamoto K, Goodyear LJ, Iannacone M, Accili D, Cantley LC, Kahn CR. Reduced expression of the murine p85alpha subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. J Clin Invest. 2002 Jan;109(1):141-9. 3. Ueki K, Fruman DA, Brachmann SM, Tseng YH, Cantley LC, Kahn CR. Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival. Mol Cell Biol. 2002 Feb;22(3):965-77. 4. Fruman DA, Mauvais-Jarvis F, Pollard DA, Yballe CM, Brazil D, Bronson RT, Kahn CR, Cantley LC. Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha. Nat Genet. 2000 Nov;26(3):379-82. 5. Hallmann D, Trumper K, Trusheim H, Ueki K, Kahn CR, Cantley LC, Fruman DA, Horsch D. Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha. J Biol Chem. 2003 Feb 14;278(7):5099-108. Epub 2002 Nov 14. 6. Ueki K, Yballe CM, Brachmann SM, Vicent D, Watt JM, Kahn CR, Cantley LC. Increased insulin sensitivity in mice lacking p85beta subunit of phosphoinositide 3-kinase. Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):419-24. 7. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 2002 Jul;10(1):151-62. 8. Chang JD, Field SJ, Rameh LE, Carpenter CL, Cantley LC. Identification and characterization of a phosphoinositide phosphate kinase homolog. J Biol Chem. 2004;279:11672-9. 9. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley LC. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A. 2004;101:3329-35.