Human phosphoinositide 3-kinase C2beta, the role of calcium and the C2 domain in enzyme activity.
The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2beta) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinases in vitro, PI 3-kinase C2beta utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4, 5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2beta, like the Class I PI 3-kinases, but unlike PI 3-kinase C2alpha, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2beta is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions.
Arcaro
A
A
Ludwig Institute for Cancer Research, University College, London W1P 8BT, United Kingdom.
Volinia
S
S
Zvelebil
M J
MJ
Stein
R
R
Watton
S J
SJ
Layton
M J
MJ
Gout
I
I
Ahmadi
K
K
Downward
J
J
Waterfield
M D
MD
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium