CAPS Acts at a Prefusion Step in Dense-Core Vesicle Exocytosis as a PIP2 Binding Protein

Abstract

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

“…CAPS is part of the machinery required for priming Ca 2ϩ -dependent vesicle exocytosis (2,4,6,7). The major conclusion of the current work is that phosphorylation of CAPS at Ser-5, -6, and -7 by protein kinase CK2 is essential for CAPS activity.…”

“…We determined whether CAPS phosphorylation was required for its activity. CAPS proteins are tested for activity by their ability to restore Ca 2ϩ -dependent vesicle exocytosis in permeable PC12 cells (4,18). A recombinant CAPS protein produced in insect Sf9 cells was purified and dephosphorylated with -protein phosphatase.…”

“…Preliminary in vitro studies with purified phosphatases showed that the N-terminal phosphorylation sites of CAPS were resistant to dephosphorylation by phosphatases PP1, PP2A, and PP2B/calcineurin. 3 Phosphatidylinositol 4,5-diphosphate is essential for priming evoked vesicle exocytosis (50) and serves as a co-factor for CAPS membrane recruitment and activation (4,19,51). Protein kinase CK2 was reported to be inhibited by the binding of phosphatidylinositol 4,5-diphosphate (52).…”

“…Many of the proteins that mediate the targeting, docking, priming, and Ca 2ϩ -dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1 2 (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca 2ϩ -triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2)(3)(4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8).…”

TheArabidopsisPhosphatidylinositol Phosphate 5-Kinase PIP5K3 Is a Key Regulator of Root Hair Tip Growth

“…It modulates the functions of a variety of actin regulatory proteins (Yin and Janmey, 2003;Logan and Mandato, 2006) and regulators of exocytotic machinery on the plasma membrane (Olsen et al, 2003;Grishanin et al, 2004;Aoyagi et al, 2005;Li et al, 2006) by directly interacting with its protein targets. In many cases, PtdIns(4,5)P 2 signaling pathways are tightly connected to those of small GTPases belonging to the Rho and Arf families in their upstream and downstream cascades (Santarius et al, 2006).…”