ErbB2 is a receptor tyrosine kinase whose activity in normal cells depends on dimerization with another ligand-binding ErbB receptor. In contrast, amplification of c-erbB2 in tumors results in dramatic overexpression and constitutive activation of the receptor. Breast cancer cells overexpressing ErbB2 depend on its activity for proliferation, because treatment of these cells with ErbB2-specific antagonistic antibodies or kinase inhibitors blocks tumor cells in the G 1 phase of the cell cycle. Intriguingly, loss of ErbB2 signaling is accompanied by a decrease in the phosphotyrosine content of ErbB3. On the basis of these results, it has been proposed that ErbB3 might be a partner for ErbB2 in promoting cellular transformation. To test this hypothesis and directly examine the role of the ''kinase dead'' ErbB3, we specifically ablated its expression with a designer transcription factor (E3). By infection of ErbB2-overexpressing breast cancer cells with a retrovirus expressing E3, we show that ErbB3 is an essential partner in the transformation process. Loss of functional ErbB2 or ErbB3 has similar effects on cell proliferation and cell cycle regulators. Furthermore, expression of constitutively active protein kinase B rescues the proliferative block induced as a consequence of loss of ErbB2 or ErbB3 signaling. These results demonstrate that ErbB2 overexpression and activity alone are insufficient to promote breast tumor cell division. Furthermore, we identify ErbB3's role, which is to couple active ErbB2 to the phosphatidylinositol 3-kinase͞protein kinase B pathway. Thus, the ErbB2͞ErbB3 dimer functions as an oncogenic unit to drive breast tumor cell proliferation.T he family of ErbB receptor tyrosine kinases includes four members: epidermal growth factor (EGF) receptor͞ErbB1, ErbB2, ErbB3, and ErbB4. Binding of peptides of the EGFrelated growth factor family to the extracellular domain of ErbB receptors results in the formation of homo-and heterodimers. Ligand binding induces the intrinsic receptor kinase activity, ultimately leading to stimulation of intracellular signaling cascades (1, 2). The physiological role of ErbB2, in the context of ErbB ligand signaling, is to serve as a coreceptor (3, 4). In fact, ErbB2 appears to be the preferred partner of the other ligandbound ErbBs (5, 6). The importance of heterodimer-mediated signaling in normal development is obvious from studies in genetically modified mice. This is particularly true for ErbB2͞ ErbB3 and ErbB2͞ErbB4 heterodimers. Loss of ErbB2 or ErbB3 has a similar impact on neuronal development (7), whereas loss of ErbB2 or ErbB4 has major effects on heart development (8, 9).A wealth of clinical data has demonstrated that ErbB receptor tyrosine kinases, in particular ErbB1 and ErbB2, have roles in human cancer development, thus making them attractive targets for cancer therapies (10-13). ErbB2 overexpression, generally attributable to gene amplification, occurs in 25-30% of breast cancer and correlates with shorter time to relapse and lower overall survival (1...

Protein phosphatase 2A (polycation-stimulated protein phosphatase L) was purified from porcine kidney and skeletal muscle. The 36-kDa catalytic and the 65-kDa putative regulatory (hereafter termed PR65) subunits of protein phosphatase 2A2 were separated by reverse-phase HPLC. Partial amino acid sequence data (300 residues) was obtained for PR65. Molecular cloning showed that two distinct mRNAs (termed alpha and beta) encoded the PR65 subunit. The cDNA encoding the alpha-isotype spanned 2.2 kilobases (kb) and contained an open reading frame of 1767 bases predicting a protein of 65 kDa, which was in good agreement with the size of the purified protein. The cDNAs encoding the beta-isotype contained an open reading frame of size similar to that of alpha-form but lacked an initiator ATG. Northern analysis, using RNA isolated from several human cell lines, indicated that the alpha-isotype was encoded by a mRNA of 2.4 kb that was much more abundant than the beta mRNA of 4.0 kb. Comparison of the predicted amino acid sequences of the two isotypes revealed 87% identity. The deduced protein sequences of the alpha- and beta-isotypes were found to be made up of 15 imperfect repeating units consisting of 39 amino acids. This repeating structure was conserved between species.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium 1 , 2 . Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.

The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3 untranslated regions. The cellular proteins binding to these RNA sequences (ARE uPA/uPAR ) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an ARE uPA -containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a ␤-globin reporter mRNA containing the ARE uPA . RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steadystate levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the ARE uPA , which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.