Association of CD38 with Nonmuscle Myosin Heavy Chain IIA and Lck Is Essential for the Internalization and Activation of CD38

Abstract

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca2؉ concentration ([Ca 2؉ ] i ), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8-or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca 2؉ ] i , and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.A type II transmembrane protein, CD38 possesses ADP-ribosyl cyclase and ADP-ribose hydrolase activities (1, 2). These two enzyme activities are involved in the conversion of ␤-NAD ϩ first to cADP-ribose (cADPR) 3 and then to ADP-ribose (3-5). The metabolite cADPR is known to increase the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) by release from intracellular Ca 2ϩ stores or by Ca 2ϩ influx through plasma membrane Ca 2ϩ channels in a variety of cells (6 -10). Mounting evidence indicates that cADPR synthesis by ADP-ribosyl cyclases is stimulated through cell-surface heterotrimeric G-protein-coupled receptor signaling. The receptors involved in ADP-ribosyl cyclase activation include the ␤-adrenergic (11, 12), angiotensin II (13), and muscarinic (14) receptors. Activation of ADP-ribosyl cyclase/CD38 by cGMP has been reported (15, 16), and cAMP-dependent activation of the enzyme has also been observed in artery smooth muscle cells (12) and cardiomyocytes (17). However, the molecular mechanism of ADPribosyl cyclase/CD38 activation has not been completely elucidated.The active site of CD38 is located in the extracellular domain, whereas the substrate ␤-NAD ϩ and the targets of the metabolite cADPR are present inside cell (18). This topological paradox of CD38 has been addressed and explained by demonstrating 1) the ligand-induced, vesicle-mediated internalization of CD38, which is followed by an increase in the intracellular cADPR concentration ([cADPR] i ) (19), and 2) the ␤-NAD ϩ -transporting function of connexin-43 and the cADPR-transporting function of membrane-bound CD38 or an unidentified cADPR transporter (20). Thus, the exact mo...

Generation of Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate by CD38 for Ca2+ Signaling in Interleukin-8-treated Lymphokine-activated Killer Cells

“…S5), although the exact mechanism has yet to be elucidated. Therefore, as we previously observed that IL-8-induced cADPR formation requires internalization of CD38 from the plasma membrane (40), but NAADP production is mediated by CD38 expressed in lysosomes via Ca 2ϩ release from the endoplasmic reticulum and cAMP/Epac/PKA/Rap 1 signaling.…”

“…S6) suggests that 2ϩ levels results in elevation of cGMP levels via guanylyl cyclase (GC), thus activating protein kinase G (PKG) (24). Phosphorylated nonmuscle myosin IIA (MHCIIA) by protein kinase G is recruited in the CD38-Lck complex, inducing the internalization of CD38 and cADPR production (40). The cADPR produced by CD38 in the endocytic vesicles is transported via the promiscuous nucleoside/cADPR transporters, as previously described (35), or by the internalized CD38 itself, as previously proposed (61).…”

“…We have demonstrated that IL-8-induced CD38 activation in LAK cells is mediated by sequential signaling involving an IL-8 receptor, IP 3 -mediated Ca 2ϩ increase, and activation of protein kinase G (24). We have also demonstrated that protein kinase G-mediated phosphorylation of nonmuscle myosin IIA and association of phospho-nonmuscle myosin IIA with CD38 via Lck are essential for internalization and activation of CD38 (40). In this study we further explored IL-8-induced signaling in LAK cells by asking whether another important Ca 2ϩ messenger, NAADP, is formed by CD38 activation.…”

“…reported that cAMP and cGMP differentially mediate the production of cADPR and NAADP in sea urchin eggs (39), and we previously demonstrated that cGMP regulated the production of cADPR through CD38 internalization, which was induced by an increase of the association CD38 with phosphorylated nonmuscle myosin IIA by protein kinase G (24,40). Therefore, we examined whether cAMP and cGMP differentially mediate the production of cADPR and NAADP by CD38 in LAK cells.…”

The Role of CD38 in Fcγ Receptor (FcγR)-mediated Phagocytosis in Murine Macrophages

“…This internalizing event has been observed in many different cellular responses where cADPR production is shifted from the surface to inside the cell (22,23). It has been suggested that the internalization mechanism is mediated by non-muscle myosin heavy chain IIA (MHCIIA), where both CD38 and MHCIIA were found to be associated in activated lymphokine-activated killer cells (24). Phagocytosis is the mechanism of internalization used by phagocytes to internalize and degrade microorganisms, cell debris, and various particles (25).…”