Summary Background In preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown. Results We showed that a combination of cell polarity and cell–cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and Amotl2 are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs) and cell–cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, Ser176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot–Lats interaction to activate the Hippo pathway. Conclusion We propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell–cell adhesion by sequestering Amot from AJ. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates.
We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.
Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKC and PKC) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKC in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKC (KD or ⌬NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKC, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone-or hyperosmolarity-induced glucose uptake, were inhibited by KD or ⌬NKD in a dosedependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKC was ϳ50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKC mutant that lacks the pseudosubstrate domain (⌬PD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of ⌬PD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKC. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKC pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.Phosphoinositide (PI) 3-kinase, a lipid kinase composed of an SRC homology 2 (SH2) domain-containing regulatory subunit and a 110-kDa catalytic subunit, catalyzes phosphorylation of the D3 position of PIs (46,48). This enzyme was first identified complexed with SRC kinase and the middle T antigen of polyomavirus and was later found to associate with various tyrosine-phosphorylated proteins in response to stimulation of cells with growth factors or cytokines (46, 48). Activation of PI 3-kinase, either by targeting of the enzyme to the plasma membrane (27) or as a consequence of direct interaction between the SH2 domain of the regulatory subunit and phosphorylated tyrosine residues present within specific motifs (5), results in the triggering of various important biological actions. Thus, with the use of either a dominant negative protein that blocks the interaction between PI 3-kinase and tyrosine-phosphorylated proteins (21,33,41) or pharmacological inhibitors of the enzyme, such as wortmannin or LY294002 (...
In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKCλ, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKCλ knockout mice, neuroepithelial cells of the neocortical region lost aPKCλ protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKCλ is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKCλ knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.
Overexpression of a TPA‐insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet‐derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal‐dependent changes in aKPClambda were observed for a PDGF receptor add‐back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3‐kinase, and were inhibited by wortmannin, an inhibitor of PI3‐kinase. Furthermore, an N‐terminal fragment of the catalytic subunit of PI3‐kinase, p110alpha, inhibited aPKClambda‐dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3‐kinase.
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