CRB3 and the FERM protein EPB41L4B regulate proliferation of mammary epithelial cells through the release of amphiregulin.

Numerous observations have suggested a connection between the maintenance of cell polarity and control of cell proliferation; however, the mechanisms underlying these connections remain poorly understood. Here we found that ectopic expression of CRB3, which was previously shown to restore tight junctions and membrane polarity in MCF-10A cells, induced a hyperproliferative phenotype, with significantly enlarged acini in basement membrane culture, similar to structures induced by expression of proliferative oncogenes such as cyclinD1. We found that CRB3-induced proliferation is epidermal growth factor (EGF)-independent and occurs through a mechanism that involves secretion of the EGF-family ligand, amphiregulin (AREG). The increase in AREG secretion is associated with an increase in the number and size of both early and late endosomes. Both the proliferative and endocytic phenotypes associated with CRB3 expression require the FERM-binding domain (FBD) but not the PDZ-binding domain of CRB3, arguing that this proliferative phenotype is independent of the PDZ-dependent polarity signaling by CRB3. We identified the FBD-containing protein, EPB41L4B, as an essential mediator of CRB3-driven proliferation and observed that the CRB3-dependent changes in endocytic trafficking were also dependent on EPB41L4B. Taken together, these data reveal a previously uncharacterized role for CRB3 in regulating proliferation in mammalian cells that is associated with changes in the endocytic trafficking machinery.

Measurement of G protein-coupled receptor-stimulated phospholipase D activity in intact cells.

Mammalian phospholipase D (PLD) activity hydrolyzes phosphatidylcholine (PC) into phosphatidic acid (PA) and free choline. This activity can be stimulated by a wide variety of extracellular agonists, including those for G protein-coupled receptors (GPCRs). This chapter outlines a protocol for the measurement of PLD activity in intact cells following stimulation by an extracellular agonist. The protocol takes advantage of a unique property of mammalian PLDs--the ability to substitute a primary alcohol for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of a phosphatidylalcohol, which is a specific and unique marker for PLD activity. This protocol is highly sensitive for the detection of PLD activity following the stimulation of intact cells, being a valuable method for studying the regulation of PLD activity in vivo.

PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1.

BACKGROUND: The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation via the downstream targets ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). We have identified phosphatidic acid (PA) as a mediator of mitogenic activation of mTOR signaling. In this study, we set out to test the hypotheses that phospholipase D 1 (PLD1) is an upstream regulator of mTOR and that the previously reported S6K1 activation by Cdc42 is mediated by PLD1. RESULTS: Overexpression of wild-type PLD1 increased S6K1 activity in serum-stimulated cells, whereas a catalytically inactive PLD1 exerted a dominant-negative effect on S6K1. More importantly, eliminating endogenous PLD1 by RNAi led to drastic inhibition of serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation in both HEK293 and COS-7 cells. Knockdown of PLD1 also resulted in reduced cell size, suggesting a critical role for PLD1 in cell growth control. Using a rapamycin-resistant S6K1 mutant, Cdc42's action was demonstrated to be through the mTOR pathway. When Cdc42 was mutated in a region specifically required for PLD1 activation, its ability to activate S6K1 in the presence of serum was hindered. However, when exogenous PA was used as a stimulus, the PLD1-inactive Cdc42 mutant behaved similarly to the wild-type protein. CONCLUSIONS: Our observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA.

Akt activation disrupts mammary acinar architecture and enhances proliferation in an mTOR-dependent manner.

Activation of the serine/threonine kinase Akt/PKB positively impacts on three cellular processes relevant to tumor progression: proliferation, survival, and cell size/growth. Using a three-dimensional culture model of MCF-10A mammary cells, we have examined how Akt influences the morphogenesis of polarized epithelial structures. Activation of a conditionally active variant of Akt elicits large, misshapen structures, which primarily arise from the combined effects of Akt on proliferation and cell size. Importantly, Akt activation amplifies proliferation during the early stages of morphogenesis, but cannot overcome signals suppressing proliferation in late-stage cultures. Akt also cooperates with oncoproteins such as cyclin D1 or HPV E7 to promote proliferation and morphogenesis in the absence of growth factors. Pharmacological inhibition of the Akt effector, mammalian target of rapamycin (mTOR), with rapamycin prevents the morphological disruption elicited by Akt activation, including its effect on cell size and number, and the cooperative effect of Akt on oncogene-driven proliferation, indicating that mTOR function is required for the multiple biological effects of Akt activation during morphogenesis.

Specificity of Rho insert-mediated activation of phospholipase D1.

Members of the Rho subfamily of GTP-binding proteins regulate phospholipase D1 (PLD1) activity and signaling. In previous work, we demonstrated that binding of the Rho family member Cdc42 to PLD1 and the subsequent stimulation of its enzymatic activity are distinct events. Deletion of the insert helix from Cdc42 does not interfere with its switch I-mediated, GTP-dependent binding to PLD1 but inhibits Cdc42-stimulated PLD1 activity. To understand the mechanism of the insert-mediated activation of PLD1 by Cdc42 and to develop reagents to study Cdc42-activated PLD1 in cellular signaling events, we have undertaken a mutational analysis of the Rho insert region of Cdc42 and examined the specificity of the insert helix requirement in the other Rho family members, RhoA and Rac1. Here, we identify a critical residue, serine 124, in the Cdc42 insert helix central to its activation mechanism. Further, we examine this activation mechanism with respect to other members of the Rho family and demonstrate that each Rho protein activates PLD by distinct mechanisms, potentially allowing for unique signaling outcomes in the cell.