myo-Inositol Monophosphatase Is an Activated Target of Calbindin D28k

Abstract

Calbindin D 28k (calbindin) is a member of the calmodulin superfamily of Ca 2؉ -binding proteins. An intracellular target of calbindin was discovered using bacteriophage display. Human recombinant calbindin was immobilized on magnetic beads and used in affinity purification of phage-displayed peptides from a random 12-mer peptide library. One sequence, SYSSIAKYPSHS, was strongly selected both in the presence of Mg 2؉ and in the presence of Ca 2؉ . Homology search against the protein sequence data base identified a closely similar sequence, ISSIKEKYPSHS, at residues 55-66 in myo-inositol-1(or 4)-monophosphatase (IMPase, EC 3.1.3.25), which constitute a strongly conserved and exposed region in the three-dimensional structure. IMPase is a key enzyme in the regulation of the activity of the phosphatidylinositol-signaling pathway. It catalyzes the hydrolysis of myo-inositol-1(or 4)-monophosphate to form free myo-inositol, maintaining a supply that represents the precursor for inositol phospholipid second messenger signaling systems. Fluorescence spectroscopy showed that isolated calbindin and IMPase interact with an apparent equilibrium dissociation constant, K D , of 0.9 M. Both apo and Ca 2؉ -bound calbindin was found to activate IMPase up to 250-fold, depending on the pH and substrate concentration. The activation is most pronounced at conditions that otherwise lead to a very low activity of IMPase, i.e. at reduced pH and at low substrate concentration.

Calbindin D28k targets myo -inositol monophosphatase in spines and dendrites of cerebellar Purkinje neurons

“…Their in vitro studies indicated that CB undergoes conformational changes upon Ca 2ϩ binding (8)(9)(10) and that CB interacts with myo-inositol monophosphatase (IMPase; ref. 7) and Ran-binding protein M (11). Together with older reports on enzyme regulation (12)(13)(14)(15) and membrane͞cytoskeleton targeting of CB (16,17), these data suggest that CB not only acts as a potent Ca 2ϩ buffer but also as a Ca 2ϩ sensor.…”

“…In recent years, evidence showing that CB may be more than a simple Ca 2ϩ buffer has been accumulated by Berggård, Linse, and coworkers (7)(8)(9) and Kumar, Cavanagh, and coworkers (10,11). Their in vitro studies indicated that CB undergoes conformational changes upon Ca 2ϩ binding (8)(9)(10) and that CB interacts with myo-inositol monophosphatase (IMPase; ref.…”

“…7), or a scrambled control peptide, SIKHPSKISEYS, were provided by the Interdisciplinary Center for Clinical Research, Leipzig, Germany or a commercial supplier (AnaSpec, San Jose, CA). Identical results were obtained with peptides from both sources.…”

Cytosolic Ca2+ Buffers

“…Although principally considered as a freely mobile protein, CB-D28k binds to several identified target proteins including Ran-binding protein (RanBP) M (Lutz et al 2003); caspase-3 (Bellido et al 2000); 3 0 ,5 0 -cyclic nucleotide phosphodiesterase (Reisner et al 1992); plasma membrane ATPase (Morgan et al 1986); L-type Ca 2þ channel a subunit (CANAC1C) (Christakos et al 2007); myo-inositol monophosphatase (IMPase) (Berggard et al 2002b); and in the kidney to TRPV5 (Lambers et al 2006). In most cases, binding studies were performed in vitro; the physiological implications of these interactions are not clear yet.…”

A Sensitive Period of Mice Inhibitory System to Neonatal GABA Enhancement by Vigabatrin is Brain Region Dependent

“…The increase in CB levels in the hippocampi of GVG-treated newborn and adult mice, without any corresponding alteration in the density or size of CB-IR neurons, might indicate an increase in cellular levels of this protein. The CB protein serves as an endogenous fast Ca 2 + buffer (Nägerl et al, 2000) and thus it regulates activity-dependent processes in synapses (Blatow et al, 2003;Schmidt et al, 2005) and protein activation (Berggard et al, 2002). Therefore, modified cellular CB levels in GVG-treated mice may result in a change in synaptic strength, excitability, and modulation of signaling pathways involving Ca 2 + .…”