Inositol 1,4,5-Trisphosphate 3-Kinase-A Is a New Cell Motility-promoting Protein That Increases the Metastatic Potential of Tumor Cells by Two Functional Activities

Abstract

The formation of distant metastases is a complex process involving escape of cancer cells from the primary tumor, dissemination to distant organs, and finally re-colonization and expansion (1). For metastatic dissemination, the cancer cell must acquire the ability to migrate, which is associated with cytoskeletal re-arrangements. Migrating cells extend actinbased filopodia and lamellipodia at the leading edge. To initiate this process, a local formation of F-actin is required, which can be mediated by actin nucleating (e.g. Arp2/3 and formins (2)), F-actin bundling (e.g. fascin (3)), and by F-actin cross-linking proteins (e.g. filamins (4)). The actin-severing proteins ADF/ cofilin and gelsolin depolymerize F-actin and thus increase actin turnover (5). The balance between stimulation of actinpolymerizing proteins and actin-depolymerizing proteins is tightly regulated by distinct signaling pathways (e.g. phospholipase C and phosphoinositide 3-kinase (6 -8)). As activation of these pathways can also result in induction of proliferation, "fine-tuning" of continuous signal inputs determines the cellular response. This fine-tuning is mediated by various small molecules, including calcium, cyclic AMP, phosphatidylinositol phosphates, and inositol phosphates. Among the inositides, membranous phosphatidylinositol 4,5-bisphosphate plays a central role in the control of migration as it regulates the activity of cofilin, gelsolin, and profilin (9, 10) and serves as a substrate for the production of the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ).2 Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate increases Ins(1,4,5)P 3 levels and subsequent calcium release from the endoplasmic reticulum. Calcium plays an important role in cell migration because calcium transients activate gelsolin and indirectly ADF/cofilin (9, 10).Inositol 1,4,5-trisphosphate 3-kinase isoenzymes (ITPKA, ITPKB, and ITPKC) metabolize Ins(1,4,5)P 3 to inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ), and thus regulate Ins(1,4,5)P 3 -induced calcium signals (11). The ITPK isoenzymes are highly conserved in their catalytically active C-terminal domains but show large differences in their N-terminal regulatory domains mediating mainly cellular targeting. The isoenzymes differ in subcellular localization and tissue expression patterns. ITPKB and ITPKC mRNAs are ubiquitously expressed, whereas mRNA of ITPKA was only identified in neurons and testis (12). In neurons, ITPKA was shown to be targeted to F-actin via an N-terminal actin binding domain (amino acids 1-66 (13)) and was suggested to be relevant for long term potentiation and spatial learning (14,15

Human Inositol 1,4,5-Trisphosphate 3-Kinase Isoform B (IP3KB) Is a Nucleocytoplasmic Shuttling Protein Specifically Enriched at Cortical Actin Filaments and at Invaginations of the Nuclear Envelope

“…This would counteract a shortening of the Ins(1,4,5)P 3 transient by the above mechanism. Recently, it was discussed that this inhibitory effect mediates IP3KA-regulated activation of store-operated Ca 2ϩ entry (42) and therefore may prolong the Ca 2ϩ release induced by Ins(1,4,5)P 3 .…”

“…An additional increase in Ca 2ϩ ϳ360 s after carbachol stimulation was observed in one-half of the cells. Possibly, the moderate elevation of Ins(1,3,4,5)P 4 caused by overexpression of the IP3KB mutant protects Ins(1,4,5)P 3 by inhibiting Ins(1,4,5)P 3 5-phosphatase (42). This protection mechanism may become increasingly important with time after stimulation because the amount of Ins(1,4,5)P 3 newly released from the limited pool of phosphatidylinositol 4,5-bisphosphate, decreases.…”

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

“…The same protocol as described 6 was used. Expression of ITPKA was evaluated with two different antibodies: with a polyclonal antibody from goat (Sc-11206 Lot#191,Santa Cruz, CA) and with a polyclonal antibody from rabbit (14270-1-AP, Proteintech, Chicago, IL), both antibodies were used in a dilution of 1:2000.…”

“…Immunohistochemistry was essentially performed as described, 6 with the following exceptions: The primary polyclonal anti-ITPKA antibody was from rabbit (14270-1-AP, Proteintech, Chicago, IL). It was diluted 1:200 and incubated with the tissue section over night at 4 C. The secondary antibody and the acetylethylcarbazol chromogen system were used according to the manufacturer's instructions (R&D Systems Rabbit-Kit, McKinley, Minneapolis).…”

“…We revealed that particular tumor cell lines express ITPKA and demonstrated that this ectopic expression of ITPKA increased the metastatic potential of tumor cells. Because of its F-actin bundling activity, ITPKA induces the formation of filopodia-and lamellipodia-like protrusions, 5,6 which are critical for cells to migrate. 7 This nonenzymatic migration-promoting effect of ITPKA is stimulated by its enzyme activity in response to stimulation with growth factors.…”