Purification and properties of d-myo-inositol 1,4,5-trisphosphate 3-kinase from bovine iris sphincter smooth muscle: effects of protein phosphorylation in vitro and in intact muscle

Abstract

Stimulation of bovine iris sphincter muscle with carbachol (10 microM) increased accumulation of Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) by 86 and 32% respectively. Addition of isoproterenol (5 microM) to muscle pretreated with carbachol reduced the 3H-radioactivity in InsP3 by 30% and increased that of InsP4 by 41%. InsP3 3-kinase was predominantly localized in the soluble fraction (110,000 g supernatant) of the iris sphincter. The enzyme was purified from this fraction by sequential chromatography on DEAE-cellulose, calmodulin (CAM)-agarose affinity, and Mono-Q anion-exchange columns. The specific activity of the purified enzyme was 1.94 mumol/min per mg protein with a purification of 114-fold, compared with the cytosolic fraction of the muscle. SDS/PAGE showed the enzyme to be associated with a protein band corresponding to 50 kDa. In the presence of 10 microM Ca2+, CaM dose-dependently stimulated the enzyme. InsP3 3-kinase specifically phosphorylated InsP3 with an apparent K(m) of 0.56 microM and a Vmax. of 2.5 mumol/min per mg protein. The stimulatory effect of CaM was due to a change in Vmax. and not in its K(m). The enzyme was maximally active at pH 7.0-7.5. Phosphorylation of the purified InsP3 3-kinase with protein kinase A increased its activity; in contrast, phosphorylation with protein kinase C inhibited the enzyme activity. Treatment of the intact iris sphincter with isoproterenol or phorbol 12,13-dibutyrate resulted in stimulation of InsP3 3-kinase activity in the soluble fraction and this activation was preserved on SDS/PAGE and renaturation. These results indicate that the bovine iris sphincter contains a Ca-CaM-dependent InsP3 3-kinase which can be differentially regulated, both in vitro and in intact muscle, by protein kinases A and C.

Membrane association, localization and topology of rat inositol 1,4,5-trisphosphate 3-kinase B: implications for membrane traffic and Ca2+ homoeostasis

“…Also, these studies do not address the possibility of a membrane-associated activity in the tissues or cell lines examined. When membrane fractions have been analysed, IP $ 3-kinase activity has been detected in turkey erythrocytes [54] and bovine iris sphincter smooth muscle [55]. Taken together, these data present strong evidence in favour of an IP $ 3-kinase activity being present in intracellular membranes.…”

Expression, Purification, and Regulation of Two Isoforms of the Inositol 1,4,5-Trisphosphate 3-Kinase

“…Regulation of IP3K Activity by Phosphorylation-In addition to stimulation by calcium/calmodulin, phosphorylation is another well documented mode of regulating IP3K activity (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). To compare the effect of phosphorylation on the activity of the two isoforms of IP3K, the purified, recombinant proteins were phosphorylated in vitro with pure PKA and PKC and examined for the stoichiometry of phosphorylation and changes in activity.…”

“…The IP3K is also a substrate for the cyclic AMP-dependent protein kinase, the calcium/calmodulin-dependent protein kinase II, and protein kinase C in vitro. When the IP3K purified from rat brain or bovine smooth muscle is phosphorylated by the cyclic AMP-dependent protein kinase in vitro, its activity is increased about 2-fold (17,18) while phosphorylation of the protein by protein kinase C reduced activity to about 25% of the basal activity (17)(18)(19). Phosphorylation of IP3K from brain with the calcium/calmodulindependent protein kinase II increases its V max about 9-fold and decreases the K m for calmodulin from 52 to 2 nM (20).…”

Arabidopsis Inositol Polyphosphate 6-/3-Kinase Is a Nuclear Protein That Complements a Yeast Mutant Lacking a Functional ArgR-Mcm1 Transcription Complex

“…Interestingly, the recently characterized Ins(1,4,5)P 3 kinase from the nematode Caenorhabditis elegans lacks the consensus calmodulin binding site and, not surprisingly, is insensitive to Ca 2 ϩ /calmodulin (Clandinin et al, 1998). Phosphorylation experiments demonstrated that Ins(1,4,5)P 3 kinase isoforms A and B are substrates for protein kinase C-and protein kinase A-mediated phosphorylation in vitro Wang et al, 1995;Communi et al, 1999).…”