Neuronal IP₃3-Kinase is an F-actin–bundling Protein: Role in Dendritic Targeting and Regulation of Spine Morphology

Abstract

The actin microstructure in dendritic spines is involved in synaptic plasticity. Inositol trisphosphate 3-kinase A (ITPKA) terminates Ins(1,4,5)P(3) signals emanating from spines and also binds filamentous actin (F-actin) through its amino terminal region (amino acids 1-66, N66). Here we investigated how ITPKA, independent of its kinase activity, regulates dendritic spine F-actin microstructure. We show that the N66 region of the protein mediates F-actin bundling. An N66 fusion protein bundled F-actin in vitro, and the bundling involved N66 dimerization. By mutagenesis we identified a point mutation in a predicted helical region that eliminated both F-actin binding and bundling, rendering the enzyme cytosolic. A fusion protein containing a minimal helical region (amino acids 9-52, N9-52) bound F-actin in vitro and in cells, but had lower affinity. In hippocampal neurons, GFP-tagged N66 expression was highly polarized, with targeting of the enzyme predominantly to spines. By contrast, N9-52-GFP expression occurred in actin-rich structures in dendrites and growth cones. Expression of N66-GFP tripled the length of dendritic protrusions, induced longer dendritic spine necks, and induced polarized actin motility in time-lapse assays. These results suggest that, in addition to its ability to regulate intracellular Ca(2+) via Ins(1,4,5)P(3) metabolism, ITPKA regulates structural plasticity.

Functional role of inositol‐1,4,5‐trisphosphate‐3‐kinase‐A for motility of malignant transformed cells

“…Because of the fact that filopodia and lamellipodia are crucial for cells to migrate, 7 we suggest that the stimulating effect of ITPKA on cell migration and ITPKA induced formation of cell processes are functionally related. The mechanism how ITPKA induces the formation of linear filopodialike protrusions can be explained by its F-actin bundling activity 4,6 as filopodia mainly consist of linear F-actinbundles. These thin finger-like structures are required for cells to adhere to the substrate, providing the first step for cells to migrate.…”

“…As branched cell processes consist of a dense network of branched and/or crossed-linked actin filaments and ITPKA was identified as an F-actin bundling protein, 4,6 known to produce linear actin filaments, we assumed that interaction of ITPKA with additional accessory proteins may be necessary to induce the formation of lamellipodia-like protrusions. To identify such potential interaction partners, a yeast two hybrid screen was performed using a human neuronal cDNA library.…”

“…In order to specify the type of interaction, the wild type EGFP-ITPKA fusion protein, an EGFP-ITPKA fusion protein with a point mutation that prevents actin binding (L34P 4,6 ), an EGFP-ITPKA fusion protein lacking the actin binding domain (ITPKADABD, with deletion of aa 1-66 2 ), and EGFP as control were overexpressed in LN 4323 cells. As shown in Fig.…”

“…It bundles actin filaments by formation of homodimers and binding to F-actin, making ITPKA to an important regulator of spine morphology. 4 Beside this physiological role, we recently have found a pathological function of ITPKA in tumor cells. We revealed that particular tumor cell lines express ITPKA and demonstrated that this ectopic expression of ITPKA increased the metastatic potential of tumor cells.…”

Myoinositol improves sperm parameters and serum reproductive hormones in patients with idiopathic infertility: a prospective double-blind randomized placebo-controlled study

“…Therefore, spermatozoa ability to fertilize is tightly associated to a remodeling in cytoskeletal compounds. It is well-known that inositols are involved in cytoskeletal reorganization and in cellular migration (Le Clainche & Carlier, 2008;Windhorst et al, 2008;Johnson & Schell, 2009), and it is likely that MI could ameliorate the fertility rate of the patients with idiopathic infertility acting both on spermatozoa cytoskeleton rearrangements and on their migratory ability.…”