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

So-Young Rah,Sang-Jin Kim,Tae-Sik Nam,Mie-Jae Im,Hyuntae Kim,Kwang-Hyun Park,Uh-Hyun Kim

doi:10.1074/jbc.m609478200

So-Young Rah, Sang-Jin Kim + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m609478200

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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 Ca(2+) 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.

Highlights

The active site of CD38 is located in the extracellular domain, whereas the substrate ␤-NADϩ and the targets of the metabolite cADPR are present inside cell [18]
myosin heavy chain IIA (MHCIIA) Is Involved in CD38 Internalization—Because the above data indicate that complex formation of CD38 with MHCIIA and Lck activates CD38 and because MHCII is involved in protein trafficking in the process of vesicle budding [30, 31], localization of these three molecules in lymphokine-activated killer (LAK) cells was examined in the presence of IL8 with and without pretreatment with the MHCIIA inhibitor blebbistatin
We demonstrated previously that CD38 in LAK cells is activated through sequential signaling involving the IL8 receptor, inositol 1,4,5-trisphosphate-mediated increase in Ca2ϩ, and activation of protein kinase G (PKG) [16]

Summary

Introduction

The active site of CD38 is located in the extracellular domain, whereas the substrate ␤-NADϩ and the targets of the metabolite cADPR are present inside cell [18]. To identify the protein(s) located between CD38 and PKG, LAK cells metabolically labeled with [32P]orthophosphate were treated with IL8 in the absence or presence of a PKG inhibitor, (Rp)-8-pCPT-cGMP-S, and the cell extracts were subjected to immunoprecipitation using anti-CD38 mAb. treatment with IL8 significantly increased the phosphorylation level of an ϳ200-kDa protein compared with the control.

Results

Conclusion