Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain.
The ability of three pure types of bovine brain phospholipase C (PLC) and one pure rat liver PLC to utilize as substrates the recently discovered phosphatidylinositol 3-phosphate (PI-3-P), a putative phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3) was investigated. PI-3-P, PI-3,4-P2, and PIP3 are the products of phosphorylation of PI, PI-4-P, and PI-4,5-P2, respectively, by phosphoinositide 3-kinase activities that are associated with certain protein-tyrosine kinases. Although these new phospholipids have been found in intact cells, PI-3,4-P2 and PIP3 appear only after stimulation of quiescent cells with growth factors such as platelet-derived growth factor (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175) and after transformation by certain oncoproteins (L. A. Serunian, K. R. Auger, T. M. Roberts, and L. C. Cantley, manuscript in preparation). Mixtures of [3H]PI-4-P plus [32P]PI-3-P or [3H]PI-4,5-P2 plus [32P]PI-3,4-P2 or PIP3 alone were used as substrates for PLCs in vitro. After incubation with enzyme followed by extraction with chloroform/methanol/HCl, the ratio of 3H/32P in the aqueous layer revealed the selective hydrolysis of PI-4-P and PI-4,5-P2 over PI-3-P and PI-3,4-P2. High performance liquid chromatography analysis of the aqueous layer containing reaction products confirmed that only PI-4-P and PI-4,5-P2, were hydrolyzed to inositol 1,4-P2 and inositol 1,4,5-P3, respectively. These findings suggest that the turnover of PI-3-P, PI-3,4-P2, and PIP3 occurs independently of the turnover of PI-4-P and PI-4,5-P2.
Serunian
L A
LA
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Haber
M T
MT
Fukui
T
T
Kim
J W
JW
Rhee
S G
SG
Lowenstein
J M
JM
Cantley
L C
LC
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism*; D010743:Phospholipids / Q000302:isolation & purification / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010743:Phospholipids; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D010738:Type C Phospholipases