UNCORRECTED PROOF 2 Regulation of the phosphorylation of the inositol 3 1,4,5-trisphosphate receptor by protein kinase C 4 Elke Vermassen, a Rafael A. Fissore, b Nael Nadif Kasri, a Veerle Vanderheyden, a 5 Geert Callewaert, a Ludwig Missiaen, a Jan B. Parys, a, * and Humbert De Smedt a 6 a Laboratory of Physiology, K.U. Leuven Campus Gasthuisberg O/N, B-3000 Leuven, Belgium 7 b Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA 8 Received 5 May 2004 9 Available online 10 Abstract 11 The various inositol 1,4,5-trisphosphate receptor (IP 3 R) isoforms are potential substrates for several protein kinases. We 12 compared the in vitro phosphorylation of purified IP 3 R1 and IP 3 R3 by the catalytic subunit of protein kinase C (PKC). Phos- 13 phorylation of IP 3 R1 by PKC was about eight times stronger than that of IP 3 R3 under identical conditions. Protein kinase A 14 strongly stimulated the PKC-induced phosphorylation of IP 3 R1. In contrast, Ca 2þ inhibited its phosphorylation (IC 50 6 2 lM) and 15 this inhibition was further potentiated by calmodulin (CaM), while the Ca 2þ -independent CaM mutant CaM 1234 was ineffective. 16 Ca 2þ and CaM, however, did not inhibit IP 3 R3 phosphorylation by PKC. Taken together, these findings show that Ca 2þ and CaM 17 differentially regulate the PKC-mediated phosphorylation of IP 3 R1 and IP 3 R3 and are indicative for a role for the inhibition of 18 IP 3 R1 phosphorylation by Ca 2þ and CaM in the negative slope of the bell-shaped effect of Ca 2þ on IP 3 R function. 19 Ó 2004 Published by Elsevier Inc. 20 Keywords: Inositol 1,4,5-trisphosphate receptor; Protein kinase C; Calmodulin; Bell-shaped Ca 2þ dependence 21 Stimulation of cells by hormones, neurotransmitters, 22 and growth factors leads in most cells to the activation 23 of phospholipase C and the subsequent production of 24 inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. 25 Diacylglycerol and Ca 2þ activate protein kinase C 26 (PKC), while IP 3 diffuses into the cell and activates the 27 IP 3 receptor (IP 3 R), an intracellular Ca 2þ -release chan- 28 nel. In mammalian cells three different IP 3 R isoforms 29 are expressed, which are differentially affected by IP 3 , 30 Ca 2þ , ATP, and accessory proteins [1,2] such as, e.g., 31 calmodulin (CaM) [3,4]. This intricate regulation of the 32 different IP 3 R isoforms may explain the formation of 33 complex, cell-specific spatio-temporal Ca 2þ signals that 34 are generated by IP 3 -dependent Ca 2þ release from in- 35 tracellular Ca 2þ stores [5]. Recently, it has become in- 36 creasingly apparent that the phosphorylation of IP 3 Rs 37 could also play an important role in the generation and 38 regulation of IP 3 -induced Ca 2þ signals. IP 3 Rs contain 39 multiple consensus sites for protein kinase-mediated 40 phosphorylation [1] and several protein kinases have 41 already been shown to phosphorylate IP 3 Rs [6]. The 42 type-1 IP 3 R (IP 3 R1) has been reported to be phos- 43 phorylated by protein kinase A (PKA) [7], protein ki- 44 nase G (PKG) [8,9], PKC [10], Ca 2þ /CaM-dependent 45 kinase II (CaMKII) [7], the tyrosine kinases Fyn [11], 46 and Lyn [12], Rho kinase [13], and cdc2/cycline B [14]. 47 The other IP 3 R isoforms (IP 3 R2 and IP 3 R3) also con- 48 tain multiple consensus phosphorylation sites [1,6], but 49 their effective phosphorylation has only been demon- 50 strated for PKA [15] and, more recently, for Rho kinase 51 [13]. 52 Since the activation of phospholipase C produces 53 both diacylglycerol and IP 3 that, respectively, trigger the 54 activation of PKC and IP 3 R [16], the role of PKC in the 55 modulation of IP 3 -induced Ca 2þ signalling is particu- 56 larly interesting. PKC has been shown to indirectly af- 57 fect Ca 2þ signalling by affecting either upstream 58 components of the IP 3 /Ca 2þ -signalling pathway such as 59 G-protein coupled receptors or phospholipase C [17] or * Corresponding author. Fax: +32-16-345991. E-mail address: jan.parys@med.kuleuven.ac.be (J.B. Parys). 0006-291X/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.05.071 Biochemical and Biophysical Research Communications 319 (2004) 888–893 BBRC www.elsevier.com/locate/ybbrc YBBRC 11450 DISK / 21/5/04 / Jaya(CE) / Anitha(TE) No. of pages: 6 DTD 4.3.1 / SPS ARTICLE IN PRESS