Antibody Cross-linking of the Glycosylphosphatidylinositol-linked Protein CD59 on Hematopoietic Cells Induces Signaling Pathways Resembling Activation by Complement

Abstract

CD59 is a glycosylphosphatidylinositol-anchored cell surface glycoprotein involved in protecting cells from host-mediated complement attack. Studies have shown that antibody cross-linking of CD59 induces a series of intracellular signaling events including the activation of protein-tyrosine kinases (PTK). To further characterize these events, antibodies and complement 8, one of the natural ligands of CD59, were used to activate CD59. Antibody-induced cross-linking of CD59 on the surface of THP-1 and U937 hematopoietic cell lines as well as exposure to complement 8 induces a rapid increase in the tyrosine phosphorylation of several proteins within the cell. Consistent with an early role for the Src family PTKs in these signaling events, we found that transient activation of Hck-and CD59-mediated signaling was abrogated in the presence of the Src family PTK-selective inhibitor PP1. Although the molecular mechanism by which CD59 communicates to Hck is unknown, cellular fractionation studies indicated that both CD59 and Hck are compartmentalized in plasma membrane microdomains. We also detected tyrosine phosphorylation of the adaptor proteins p120 cbl and Shc, and the cytoplasmic nonreceptor tyrosine kinase Syk. The identification of CD59-mediated signaling events may help explain why paroxysmal nocturnal hemoglobinuria patients, who are deficient in glycosylphosphatidylinositol-linked proteins including CD59, are susceptible to proliferative disorders.CD59 is one of a large family of proteins that are attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) 1 moiety attached to their C-terminal ends (1). It is abundantly expressed on a wide variety of cells, including many cells of hematopoietic lineage (2-5), and functions to inhibit complement-mediated lysis by interfering with the assembly of the membrane attack complex and the insertion of complement 9 into the cell membrane (reviewed in Ref.6). The natural ligands for CD59 include the ␣-chain of complement 8 (C8) and the "b" domain of complement 9 (7).Antibody-mediated cross-linking of CD59 on various hematopoietic cells induces a cascade of intracellular signaling activities including events such as calcium influx and release of calcium from intracellular stores, generation of reactive oxygen intermediates (8 -12), and induction of tyrosine phosphorylation (13,14). These events are not unique to CD59, since many other GPI-anchored proteins induce a series of similar intracellular signaling events when cross-linked or upon binding of appropriate ligand. Other GPI-linked molecules expressed on hematopoietic cells include the lipopolysaccharide receptor (CD14) (15, 16) and THY-1, a regulatory molecule in T-cell activation (17, 18).Since GPI-anchored proteins adhere to cells through their acyl chains and do not span the membrane, the mechanism by which this family of proteins transduce their signal into the cell remains unknown. However, GPI-linked proteins have been shown to cluster in detergent-insoluble glycolipid-enriche...

Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration

“…Signalling by a number of GPI-anchored proteins is mediated by Src kinase activation (Mouillet-Richard et al, 2000;Murray and Robbins, 1998;Wei et al, 2007). We investigated the role of Src in the Prod1 signalling cascade and found that the Src kinase inhibitor PP2 showed no inhibition of MMP9 induction when assayed by qRT-PCR (supplementary material Fig.…”

“…2A) upregulates MMP9 secretion in Prod1-transfected cells. We conclude that ERK1/2 and EGFR activation are significant components of the Prod1 response at the level of mRNA expression and subsequent protease secretion, but can only account for part of the MMP9-inducing activity.Signalling by a number of GPI-anchored proteins is mediated by Src kinase activation (Mouillet-Richard et al, 2000;Murray and Robbins, 1998;Wei et al, 2007). We investigated the role of Src in the Prod1 signalling cascade and found that the Src kinase inhibitor PP2 showed no inhibition of MMP9 induction when assayed by qRT-PCR (supplementary material Fig.…”

Further Characterization of Reproductive Abnormalities in mCd59b Knockout Mice: A Potential New Function of mCd59 in Male Reproduction

“…Indeed, Western blot analysis with specific Abs against phosphotyrosine has become an accepted method to assess sperm capacitation (29,30,34). In other cell types, CD59 has been found to be physically connected to the membrane tyrosine phosphorylation machinery; if this were the case in sperm cells, CD59 could play a role in sperm capacitation (35)(36)(37). To investigate whether the absence of mCd59 could affect capacitation-dependent tyrosine phosphorylation and ionophore-induced acrosome reaction in mouse sperm, we analyzed both tyrosine phosphorylation and acrosome reaction in sperm obtained by the swim-out method from the epididymis of 3-moold mice.…”

Complement C2 Receptor Inhibitor Trispanning: A Novel Human Complement Inhibitory Receptor

“…In this study, we have found that anti-CRIT-ed1 blockage of the CRIT receptor increases the complement-mediated killing of two human myeloid cell lines (U937 and THP-1) and of monocytes. However, the relative contribution of CRIT to protecting against complement-dependent cytotoxicity in the THP-1 cells, for example, cannot be known, because certain other complement regulatory proteins are expressed, such as CD59 (40). Once C2 is bound to CRIT (unlike C2 bound to C4b), it cannot be cleaved by C1s to yield C2a and C2b and thus no longer partakes in C3 convertase formation.…”

“…If the dimerization of CRIT occurs through the formation of intermolecular disulfide bonds then candidate cysteine residues would be Cys 31,40,41 of TM1, Cys 69 of TM2, Cys 110 of TM3, or Cys 126,214,280 of the cytoplasmic tail. Disulfide linkages are not usually found in the cytoplasm because of the reducing environment there.…”