Inositol 1,4,5-Trisphosphate 3-Kinase A Associates with F-actin and Dendritic Spines via Its N Terminus

Abstract

The consequences of the rapid 3-phosphorylation of inositol 1,4,5-trisphosphate (IP 3 ) to produce inositol 1,3,4,5-tetrakisphosphate (IP 4 ) via the action of IP 3 3-kinases involve the control of calcium signals. Using green fluorescent protein constructs of full-length and truncated IP 3 3-kinase isoform A expressed in HeLa cells, COS-7 cells, and primary neuronal cultures, we have defined a novel N-terminal 66-amino acid F-actin-binding region that localizes the kinase to dendritic spines. The region is necessary and sufficient for binding Factin and consists of a proline-rich stretch followed by a predicted ␣-helix. We also localized endogenous IP 3 3-kinase A to the dendritic spines of pyramidal neurons in primary hippocampal cultures, where it is co-localized postsynaptically with calcium/calmodulin-dependent protein kinase II. Our experiments suggest a link between inositol phosphate metabolism, calcium signaling, and the actin cytoskeleton in dendritic spines. The phosphorylation of IP 3 in dendritic spines to produce IP 4 is likely to be important for modulating the compartmentalization of calcium at synapses.Inositol 1,4,5-trisphosphate (IP 3 ) 1 signals in neurons are terminated predominantly via the action of calcium-stimulated IP 3 3-kinases (IP3kins) (1). These enzymes produce 1,3,4,5-tetrakisphosphate (IP 4 ), which does not gate IP 3 receptor calcium channels and may have signaling properties of its own (2-4). Brain has more IP3kin activity than other tissues (5), and this is due to the concentration of the A isoform in the dendrites of principal neurons, such as cerebellar Purkinje cells, hippocampal CA1 pyramidal cells, and dentate gyrus granule cells (6 -8). Targeted disruption of IP3kin A isoform (IP3kinA) produces mice with enhanced long term potentiation but normal spatial learning (9). By contrast, injecting IP 4 into pyramidal neurons prior to tetanic stimulation strengthens the long term potentiation via a mechanism that involves the enhancement of voltage-activated calcium channels by IP 4 (10).IP3kins are members of the inositol polyphosphate kinase family, which consists of proteins sharing a conserved C-terminal catalytic region (4, 11). Animal IP3kins are the only inositol polyphosphate kinases thought to be regulated by calcium, both via direct interaction with calmodulin and by phosphorylation by calmodulin-dependent kinase II (CaMKII) (12)(13)(14). The B isoform of IP3kin, which is expressed in most cells and in glia in brain (5, 15), is localized to internal membranes of the endoplasmic reticulum and Golgi apparatus, where it is poised to regulate IP 3 concentrations near calcium stores (16). In contrast, nothing is known about targeting mechanisms for isoform A in neurons.Based on purification of the bovine and rat brain enzyme, IP3kinA activity and protein have been described as cytosolic (17)(18)(19), in contrast to type I 5-phosphatase that appears to be mostly membrane-bound (20). Immunocytochemical studies employing antibodies against the purified rat brain enzyme reveale...

Regulation of innate immunity by inositol 1,3,4,5-tetrakisphosphate

“…A and B isoforms were found to be localized in both cytoskeleton and cytosolic, indicating a connection between cytoskeleton structure and InsP3K function. 5,[55][56][57] Consistent with this, similar with F-actin, InsP3KB is highly localized in the leading edge of polarized neutrophils. 16 In addition, since all three isoforms contain a calmodulin binding motif, the InsP3KB activity in neutrophils might also be regulated by calcium.…”

Synaptic Activity Augments Muscarinic Acetylcholine Receptor-stimulated Inositol 1,4,5-Trisphosphate Production to Facilitate Ca2+ Release in Hippocampal Neurons

“…1C) in the presence of 0.5 M TTx to block synaptic activity. This resulted from increased IP 3 levels as opposed to PIP 2 depletion because metabolism of IP 3 by co-expression with a DsRed2-tagged IP 3 3-kinase A, but not the kinase-dead enzyme (22,24), inhibited GFP-PH PLC␦ translocation by Ͼ80% (Fig. 1, D and E, n ϭ 11, p Ͻ 0.001).…”

“…Hippocampal Cell Culture-Rat hippocampal neurons from 1-3-dayold rat pups were grown in serum-free conditions using previously described methods (10,24). Briefly, isolated hippocampi were cut into small pieces of about 2-3 mm 3 and trypsinized for 30 min using 0.5 mg ml Ϫ1 Pronase E and 0.5 mg ml Ϫ1 thermolysin in a HEPES-buffered salt solution (HBSS) containing 130 mM NaCl, 10 mM HEPES, 5.4 mM KCl, 1 mM MgSO 4 , 25 mM glucose, and 1.8 mM CaCl 2 at pH 7.2.…”

Roles of Phosphorylation-dependent and -independent Mechanisms in the Regulation of M1 Muscarinic Acetylcholine Receptors by G Protein-coupled Receptor Kinase 2 in Hippocampal Neurons

“…Cell Culture and Transfections-Hippocampal neurons from 1-dayold Lister-hooded rat pups were isolated as described previously (22,23). Briefly, isolated hippocampi were dissociated with Pronase E (0.5 mg ml Ϫ1 ) and thermolysin (0.5 mg ml Ϫ1 ) in a HEPES-buffered salt solution (130 mM NaCl, 10 mM HEPES, 5.4 mM KCl, 1.0 mM MgSO 4 , 25 mM glucose, and 1.8 mM CaCl 2 , pH 7.2) for 30 min.…”

“…Expression-The ability of a specific antisense GRK2 construct (almost the full length of rat GRK2 cloned into pcDNA3 in an antisense direction) (23) to suppress endogenous GRK2 expression was initially determined after transfection of HEK293 cells with 1, 2, or 3 g of either pcDNA3 (control) or antisense GRK2 using Lipofectamine 2000, according to the manufacturer's instructions. After 72 h the cells were lysed, and GRK2 expression was determined by Western blotting using a rabbit polyclonal anti-GRK2 antibody (Santa Cruz).…”