CD4 Is Active as a Signaling Molecule on the Human Monocytic Cell Line Thp-1 Gina M. Graziani-Bowering,* Lionel G. Filion,* Pierre Thibault,† and Maya Kozlowski* , ‡ ,1 *Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada K1H 8M5; †Institute for Biological Sciences, National Research Council, Ottawa, Canada K1A 0R6; and ‡Biologics and Genetics Therapies Directorate, Centre for Biologics Research, Health Canada, Ottawa, Canada K1A 0L2 CD4 is a 56-kDa membrane glycoprotein expressed by a subset of T cells, by cells of the monocyte/macro- phage lineage, and by eosinophils and dendritic cells. CD4 serves as a coreceptor for HIV and IL-16. T cell CD4 mediates signal transduction by associating with the protein tyrosine kinase p56 lck ; this interaction does not exist in monocytes. We wished to elucidate the mechanism(s) by which monocyte CD4 transduces sig- nals. Stimulation of CD4 on Thp-1 monocytic cells in- duced a Ca 2 flux and the time-dependent activation of phosphotyrosine proteins ranging from 35 to 180 kDa. We identified the 140- and 85-kDa proteins as phospho- lipase C gamma (PLC-) and the regulatory subunit of phosphatidylinositol 3-kinase (PI-3K), respectively. Using immunoprecipitation/Western immunoblotting however, we were unable to show any direct associa- tion between CD4 and PLC-, PI-3K, or other known signaling proteins. To identify proteins capable of as- sociating with the cytoplasmic tail of CD4, we fused it with gluthatione S-transferase and used the fusion protein in far Western and pull-down experiments. In both types of experiments, the fusion protein rou- tinely associated with 45- and 55-kDa proteins. Mass spectrometry analysis of the tryptic peptides gener- ated from these two proteins indicated novel se- quences. © 2002 Elsevier Science (USA) Key Words: CD4; monocytes; Thp-1; signal transduc- tion; Ca 2 flux; PI3-K; PLC-1. INTRODUCTION The CD4 molecule is a 56-kDa membrane glycopro- tein [1]. In humans, CD4 is expressed by a subset of T cells [1], cells of the monocyte/macrophage lineage [2], dendritic cells [2], and eosinophils [3]. The CD4 mole- cule has been the subject of intense study in recent years given its function as a HIV coreceptor [4 – 6], in conjunction with the seven-transmembrane G-protein- coupled chemokine receptors CCR5 and CXCR4 [7–9]. CD4 is also a coreceptor [10], apparently in conjunction with these same chemokine receptors [11, 12], for the chemokine IL-16. Additional functions for CD4 have also been identified for T cell CD4, whereas very little is known about CD4 in human monocytes/macro- phages and other CD4 + cell populations. In CD4 + T cells, CD4 plays a crucial role in the signal transduction response of the cells to antigens pre- sented to them in the context of MHC class II mole- cules (reviewed in [13]). This signal is mediated by virtue of the association of CD4 with the Src-related protein tyrosine kinase p56 lck , which functions to ty- rosine-phosphorylate proteins associated with the T cell antigen receptor complex [14, 15]. The end result is the induction of the T cell proliferation, differentiation, and cytokine synthesis and release required to mount an effective immune response to the triggering antigen (reviewed in [16]). Furthermore, in T cells, HIV-in- duced apoptosis is mediated by the CD4-p56 lck complex; the downstream signaling events are unknown [17]. Such a process may contribute to the depletion of CD4 + T cells, a hallmark of AIDS pathogenesis [17]. Unlike T cell CD4, monocytic CD4 has no associated kinase activity [18 –20]. Some investigators, however, have observed CD4-mediated signaling in monocyte/ macrophages. For example, a complex inositol poly- phosphate response and a Ca 2+ flux were induced in monocytes in which CD4 and Fc receptors were co- cross-linked [21]. IL-16 stimulation of various CD4 + monocytic cell lines induces a translocation of PKC from the cytosol to the membrane [22], as well as activation of the SAPK 2 and p38 MAPK signaling path- ways [23]. Finally, gp120 of HIV can induce a CD4- 1 To whom reprint requests should be addressed at Health Can- ada, Biologics and Genetics Therapies Directorate, Centre for Bio- logics Research, Tunney’s Pasture, Ottawa, ON, Canada K1A 0L2, Postal Locator 2201C. E-mail: Maya_kozlowski@hc-sc.gc.ca 2 Abbreviations used: Ab, antibody; APS, ammonium persulfate; Ca 2+ , calcium ion; CD4 cyt , cytoplasmic tail of CD4; DAG, diacylglyc- erol; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; HCK, hematopoietic cell kinase; Hepes, N-2-hydroxethylpiperazine-N-2-ethanesulfonic acid; IL-1, interleukin 1 beta; IL-16, interleukin 16; InsP 3 , inositol 1,4,5-trisphosphate; IPTG, isopropylthio--D-galactoside; 2ME, 2-mercaptoethanol; MAPK, mito- 0014-4827/02 $35.00 141 © 2002 Elsevier Science (USA) All rights reserved. Experimental Cell Research 279, 141–152 (2002) doi:10.1006/excr.2002.5581