Signal transduction of fertilization in frog eggs and anti-apoptotic mechanism in human cancer cells: common and specific functions of membrane microdomains

Abstract

Membrane microdomains or lipid/membrane rafts are distinct areas on the plasma membranes, where a specific subset of lipids (e.g. cholesterol, sphingolipids) and proteins (e.g. glycosylphosphatidylinositol-anchored proteins, growth factor receptor/kinases) are getting together and functioning for several aspects of cellular functions. Our recent investigation has revealed that fertilization of African clawed frog, Xenopus laevis, requires cholesterol-dependent nature of egg membrane microdomains. Moreover, fertilization of Xenopus eggs involves proteolytic cleavage of the extracellular part and subsequent phosphorylation of a cytoplasmic tyrosine residue of uroplakin III, an egg membrane microdomain-associated protein. Protease activity toward uroplakin III seems to be derived from fertilizing sperm, while phosphorylation of uroplakin III seems to be catalyzed by the egg tyrosine kinase Src, whose activation is required for cytoplasmic rearrangement of fertilized eggs; so-called 'egg activation'. Therefore, it is assumed that uroplakin III serves an integral part of signal transduction in fertilization of Xenopus. Our more recent study on human cancer cells has revealed that a similar but distinct scheme of signal transduction operates in anti-apoptotic growth of cells. Namely, in human bladder carcinoma cells, cooperation of uroplakin III and Src, both of which localize to the membrane microdomains, allows cells to escape from apoptotic cell death and proliferate under culture conditions deprived of serum. In this review, I briefly introduce about biology of fertilization and cancer, and then present and discuss our experimental data on general importance and specific features of membrane microdomains in Xenopus fertilization and anti-apoptosis in human bladder carcinoma cells.

Fig. (1)

Signal transduction leading to Ca²⁺transient at fertilization in sea urchin, Xenopus laevis (frog), Cynops pyrrhogaster (newt), and mouse. Shown schematically is sequence of events at fertilization in four model organisms (sea urchin as representative of sea invertebrates, frog and newt as representative of amphibian vertebrates, and mouse as representative of mammalian vertebrates). In all species, IP3-dependent mechanism is a dominant component of intracellular Ca²⁺ release (as highlighted in red), which is necessary for successful egg activation by sperm. In most species, molecular detail of sperm-egg membrane interaction and fusion is unknown and may be varied between species (as highlighted in green). Special note that genetic approach has identified sperm Izumo and egg CD9 as essential components for sperm-egg fusion. Egg cytoplasmic events connecting sperm-egg interaction/fusion and IP3-dependent Ca²⁺ release are semi-conserved among species and involve tyrosine kinase-dependent or-independent actions of PLCs (as highlighted in yellow)

Fig. (2)

Study of fertilization signaling focusing on structure and function of egg plasma membrane microdomains. (A) Schematic drawing of preparation of membrane microdomains from Xenopus eggs, which are unfertilized, fertilized, or artificially activated (parthenogenetically activated), and its utility in functional analysis. (B) Schematic diagram showing a series of events that operates during early phase of fertili-zation of Xenopus eggs. Sperm-induced egg activation may involve proteolytic cleavage of the egg membrane microdomain-associated uro-plakin III, catalyzed by sperm-derived protease. Uroplakin III is in association with uroplakin Ib, a tetraspanin molecule that supports the localization of uroplakin III to membrane microdomains. The uroplakin III-uroplakin Ib complex is involved in negative regulation of cyto-plasmic tyrosine kinase Src, and proteolytic cleavage of uroplakin III may lead to up-regulation of Src by unknown mechanism. Activated Src phosphorylates and activates PLC,γ which in turn promotes IP3 production and Ca²⁺transient.Src may also phosphorylate intact uro-plakin III, although its physiological importance has not yet been demonstrated

Fig. (3)

Initiation, promotion and progression of carcinogenesis: from benign tumor to malignant tumor. (A) Survival and growth under microenvironment where no supply of energy and nutrient is available. Serum starvation in cell culture may mimic such a microenvironment in vitro. (B) Anti-apoptotic mechanism of cancer cells. Cell become invasive and enter newly formed capillary (induction of angiogenesis). (C) Newly formed capillary supports growth and survival of tumor. Cells travel through bloodstream and undergo metastasis. This scheme is adapted from “ The Bi9ology of Cancer” by R.A. Weinberg (Garland Science, 2007).

Fig. (4)

Anti-apoptotic mechanism of survival and growth of serum-starved 5637 human bladder carcinoma cells. Serum starvation signal may trigger both non-genomic (e.g. catalytic and/or physical protein interactions) and genomic responses (gene expression and/or protein synthesis) in 5637 cells. At early phase of signal transduction, activation of Src by unknown mechanism, activation of MT1-MMP by tyro-sine phosphorylation and/or increased protein expression, secretion of soluble HB-EGF into the culture medium, and proteolysis of uroplakin III are operating. Secreted HB-EGF binds to and activates EGFR/kinase. Src may also be activated under the control of EGFR. Src and EGFR phosphorylate β-subunit of HGFR/c-Met (p145) on tyrosine residues 1003, 1234, and 1235. These phosphotyrosine residues in β-subunit of HGFR/c-Met may be responsible for up-regulating anti-apoptotic ability of cells through PI 3-kinase-Akt pathway and/or Grb2-Sos-Ras-MAPK pathway: the former contributes to phosphorylation and inactivation of BAD, a pro-apoptotic component of Bcl-2 family, and the latter contributes to induction of Bcl-2 and Bcl-xL, both of which are anti-apoptotic components of Bcl-2 family. These components act positively or negatively on cytochrome c oxidase, which will be released from mitochondria upon mitochondria-mediated apoptosis in response to metabolically sensed death signals (i.e. serum starvation). Cytochrome c oxidase, if released, promotes sequential activation of caspase 9 and caspase 3/7, latter of which directly contributes to apoptotic cellular processes. Thus, serum-independent survival and growth of 5637 cells may involve suppression of pro-apoptotic pathway and/or activation of anti-apoptotic pathway