Membrane association, localization and topology of rat inositol 1,4,5-trisphosphate 3-kinase B: implications for membrane traffic and Ca2+ homoeostasis.
We previously reported the isolation of a rat cDNA clone encoding a protein with significant sequence homology to the B isoform of human myo-inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase B); this protein was thus designated rat IP3 3-kinase B [Thomas, Brake, Luzio, Stanley and Banting (1994) Biochim. Biophys. Acta 1220, 219-222]. However, no IP3 kinase isoform had been shown to generate the physiologically important isoform of inositol tetrakisphosphate, i.e. inositol 1,3,4,5-tetrakisphosphate. We now present direct evidence that the putative rat IP3 3-kinase B is genuinely an IP3 3-kinase. We also show that the enzyme exists both as a peripheral membrane protein tightly associated with the cytosolic face of the extended endoplasmic reticulum network, and as a cytosolic protein. Association of the IP3 3-kinase with membranes is not affected by treatment with brefeldin A, Na2CO3 (pH 11.5), 2 M NaCl, or alteration of [Ca2+]. However, treatment of isolated membranes with 4 M urea leads to dissociation of the kinase from the membrane, implying that membrane association involves specific, conformation-dependent protein-protein interactions. The fact that IP3 3-kinase B is localized exclusively to membranes of Ca2+ stores, is consistent with a model where this kinase plays a role in IP3-dependent Ca2+ release.
Soriano
S
S
Department of Biochemistry and BBSRC-funded Molecular Recognition Centre, University of Bristol, Bristol BS8 1TD, UK.
Thomas
S
S
High
S
S
Griffiths
G
G
D'santos
C
C
Cullen
P
P
Banting
G
G
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D020126:Brefeldin A; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002254:Carbonates / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003517:Cyclopentanes / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D006706:Homeostasis; D006801:Humans; D017136:Ion Transport; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014162:Transfection; D014508:Urea / Q000494:pharmacology
D002254:Carbonates; D002352:Carrier Proteins; D003517:Cyclopentanes; D018076:DNA, Complementary; D007527:Isoenzymes; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D012965:Sodium Chloride; C005686:sodium carbonate; D014508:Urea; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium