Ian G. Macara

The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42

PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. Here we describe an unexpected link between these two systems. We have identified a family of mammalian Par6 proteins that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42–GTP, with a human homologue of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell–cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3.

Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10

The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII–C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.

Unconventional Rac-GEF activity is mediated through the Dock180–ELMO complex

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180–ELMO1 interaction is required. We can also detect a trimeric ELMO1–Dock180–Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180–ELMO complex functions as an unconventional two-part exchange factor for Rac.

CED-12/ELMO, a Novel Member of the CrkII/Dock180/Rac Pathway, Is Required for Phagocytosis and Cell Migration

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Direct interaction of two polarity complexes implicated in epithelial tight junction assembly.

Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and basolateral membranes. Two evolutionarily conserved multi-protein complexes, Crumbs (Crb)–PALS1 (Stardust)–PATJ (DiscsLost) and Cdc42–Par6–Par3–atypical protein kinase C (aPKC), have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. Here we identify a biochemical and functional link between these two complexes that is mediated by Par6 and PALS1 (proteins associated with Lin7). The interaction between Par6 and PALS1 is direct, requires the amino terminus of PALS1 and the PDZ domain of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6 with a mutated PDZ domain, to the cell surface. Expression of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of PALS1, members of the Par3–Par6–aPKC complex and the tight junction marker, ZO-1. Similarly, overexpression of Par6 in MDCK cells inhibits localization of PALS1 to the tight junction. Our data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions.

Par-3 controls tight junction assembly through the Rac exchange factor Tiam1

The par (partitioning-defective) genes express a set of conserved proteins that function in polarization and asymmetric cell division. Par-3 has multiple protein-interaction domains, and associates with Par-6 and atypical protein kinase C (aPKC). In Drosophila, Par-3 is essential for epithelial cell polarization. However, its function in mammals is unclear. Here we show that depletion of Par-3 in mammalian epithelial cells profoundly disrupts tight junction assembly. Expression of a carboxy-terminal fragment plus the third PDZ domain of Par-3 partially rescues junction assembly, but neither Par-6 nor aPKC binding is required. Unexpectedly, Rac is constitutively activated in cells lacking Par-3, and the assembly of tight junctions is efficiently restored by a dominant-negative Rac mutant. The Rac exchange factor Tiam1 (ref. 7) binds directly to the carboxy-terminal region of Par-3, and knockdown of Tiam1 enhances tight junction formation in cells lacking Par-3. These results define a critical function for Par-3 in tight junction assembly, and reveal a novel mechanism through which Par-3 engages in the spatial regulation of Rac activity and establishment of epithelial polarity.

Parsing the Polarity Code

Cell polarization is used both to mediate physical fates, as, for example, in orientated cell migration, and to specify differential phenotypic fates, as in the asymmetric division of stem cells. Strikingly, the same sets of conserved proteins are used throughout the Metazoa for these purposes. The PAR proteins organize cell polarization in many contexts, and the PINS proteins control the orientation of mitosis. These proteins seem to function as components of a self-organizing network, and an important goal is to decode — or parse — the molecular language of this network.

Mammalian Pins Is a Conformational Switch that Links NuMA to Heterotrimeric G Proteins

During asymmetric cell divisions, mitotic spindles align along the axis of polarization. In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit. A mammalian Pins, called LGN, binds Galphai and also interacts through an N-terminal domain with the microtubule binding protein NuMA. During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain. Using a FRET biosensor, we find that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization. Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements. We propose that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode.

Inhibition of Nuclear Import by Protein Kinase B (Akt) Regulates the Subcellular Distribution and Activity of the Forkhead Transcription Factor AFX

ABSTRACT AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.

Organization and execution of the epithelial polarity programme

Epithelial cells require apical–basal plasma membrane polarity to carry out crucial vectorial transport functions and cytoplasmic polarity to generate different cell progenies for tissue morphogenesis. The establishment and maintenance of a polarized epithelial cell with apical, basolateral and ciliary surface domains is guided by an epithelial polarity programme (EPP) that is controlled by a network of protein and lipid regulators. The EPP is organized in response to extracellular cues and is executed through the establishment of an apical–basal axis, intercellular junctions, epithelial-specific cytoskeletal rearrangements and a polarized trafficking machinery. Recent studies have provided insight into the interactions of the EPP with the polarized trafficking machinery and how these regulate epithelial polarization and depolarization.