Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate that in response to chemoattractants three PH domain-containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

Dictyostelium: a model for regulated cell movement during morphogenesis.

Dictyostelium has played an important role in unraveling the pathways that control cell movement and chemotaxis. Recent studies have started to elucidate the pathways that control cell sorting, morphogenesis, and the establishment of spatial patterning in this system. In doing so, they provide new insights into how cell movements within a multicellular organism are regulated and the importance of pathways that are similar to those that regulate chemotaxis of cells on two-dimensional surfaces during aggregation.

A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium.

BACKGROUND: Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. RESULTS: PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. CONCLUSIONS: Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development.

Protein kinase B/Akt is activated by polyomavirus middle-T antigen via a phosphatidylinositol 3-kinase-dependent mechanism.

The middle tumor antigen (middle-T) of mouse polyomavirus is responsible for the transforming potential of this virus. Middle-T has been shown to interact with a variety of cellular proteins known to mediate mitogenic signaling, like phosphatase-2A, Src family kinases, phosphatidylinositol 3-kinase (PI 3-kinase), the adapter protein SHC, phospholipase Cgamma-1 and 14-3-3 family proteins. Association with SHC and PI 3-kinase, respectively, stimulates two independent signaling pathways that are indispensible for viral oncogenicity. SHC activates the Ras/MAPK pathway via Grb2/SOS resulting in changes in early gene expression. The downstream targets of PI 3-kinase are less well studied but seem to impinge on serum response factor (SRF) which is also involved in regulating early gene expression. Recently, the protein kinase B/Akt (PKB/Akt) has been identified as a target of PI 3-kinase in receptor tyrosine kinase signaling. Here we show that PKB/Akt is a target of wild type middle-T, but not of mutants unable to activate PI 3-kinase. These data were confirmed using inhibitors of PI 3-kinase as well as dominant-negative alleles of the catalytic subunit of this lipid kinase. In addition, mutants of PKB/Akt lacking a pleckstrin homology domain and therefore unable to bind to D3 phospatidylinositides were not activated by middle-T. Taken together these data suggest that middle-T activates PKB/Akt in a PI 3-kinase-dependent manner. Furthermore, direct association with D3 phosphatidylinositides seems to be essential for activation of PKB/Akt.

Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins.

We describe the construction of pBact-NGFP and pBact-CGFP, two expression vectors that incorporate green fluorescent protein (GFP) as a fluorescent tag at the N- or C terminus of the produced protein. When transfected into recipient cells, GFP-tagged proteins can be visualised in the living cells using standard fluorescence microscopy techniques. Using these expression vectors, we have produced GFP-tagged versions of the neuronal microtubule-associated proteins (MAP), MAP2c and Tau34, in a number of different cell types. Both GFP-MAP2c and GFP-Tau34 were fluorescent and retained their ability to bind to microtubules. The pBact-NGFP and pBact-CGFP expression vectors represent a fast and convenient way to produce fluorescently tagged polypeptides of selected sequences encoding whole proteins or fragments for the analysis of function and dynamic events in living cells.

Activation-independent nuclear translocation of mitogen activated protein kinase ERK1 mediated by thiol-modifying chemicals.

The extracellular signal-regulated kinases ERK1 and ERK2 are key mediators of mitogenic signals in most cell types. In fibroblasts, sustained activation and nuclear translocation are mandatory for S-phase induction. The events leading to activation of these kinases are well understood, whereas little is known about the mechanism of their translocation. Using indirect immunofluorescence and biochemical analysis we show that ERK1 can translocate to the nucleus in the absence of activation and phosphorylation by upstream kinases when cells are treated with thiol-modifying chemicals. We propose that these chemicals inactivate a protein contributing to the cytoplasmic localization of ERK1.

Distribution of traction forces associated with shape changes during amoeboid cell migration.

Amoeboid motility results from the cyclic repetition of shape changes leading to periodic oscillations of the cell length (motility cycle). We analyze the dominant modes of shape change and their association to the traction forces exerted on the substrate using Principal Component Analysis (PCA) of time-lapse measurements of cell shape and traction forces in migrating Dictyostelium cells. Using wild-type cells (wt) as reference, we investigated Myosin II activity by studying Myosin II heavy chain null cells (mhcA-) and Myosin II essential light chain null cells (mlcE-). We found that wt, mlcE-and mhcA- cells utilize similar modes of shape changes during their motility cycle, although these shape changes are implemented at a slower pace in Myosin II null mutants. The number of dominant modes of shape changes is surprisingly few with only four modes accounting for 75% of the variance in all cases. The three principal shape modes are dilation/elongation, bending, and bulging of the front/back. The second mode, resulting from sideways protrusion/retraction, is associated to lateral asymmetries in the cell traction forces, and is significantly less important in mhcA- cells. These results indicate that the mechanical cycle of traction stresses and cell shape changes remains remarkably similar for all cell lines but is slowed down when myosin function is lost, probably due to a reduced control on the spatial organization of the traction stresses.

Domains in middle-T antigen that cooperate in polyomavirus-mediated oncogenic transformation.

Middle-T antigen is the oncogenic protein of Polyomavirus and associates with several cellular enzymes involved in signal transduction, e.g., Src tyrosine kinases, phosphatidylinositol 3-kinase (PI 3-kinase), protein phosphatase 2A (PP2A), and Shc, an SH2 domain-containing adapter protein. We have shown earlier that middle-T is a target of a cell cycle-regulated serine/threonine-specific kinase, presumably p34cdc2. Phosphorylation of middle-T by p34cdc2 results in increased apparent M, weight of the protein on SDS-polyacrylamide gels. Two threonine residues in positions 160 and 291, respectively, were identified in the middle-T sequence as putative targets of a cyclin-dependent kinase. Replacement of threonine 160 by alanine resulted in a transformation-defective mutant protein that was still capable of forming all the complexes with cellular proteins, suggesting that additional characteristics of middle-T are required for cell transformation. In the present study we report that the defect of the T160A middle-T mutant is compensated by mutations introduced into a domain encompassing amino acids 253 to 302. In particular, mutating serine 283, a canonical phosphorylation site for a cyclin-dependent kinase, to an alanine residue rendered the T160A middle-T mutant wild type. Based on these results we suggest that cell cycle-specific phosphorylation of specific serine and threonine residues by cyclin-dependent kinases regulates middle-T function.

Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.

Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein-coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation-stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.

[Clinical and immunological studies on a patient with myasthenia gravis before and after repeated plasma exchange]. / Klinische und immunologische Untersuchungen bei einer Patientin mit Myasthenia gravis vor und nach wiederholtem Plasmaaustausch.

A 56-year-old patient with severe acquired myasthenia gravis refractory to conventional therapy underwent a series of plasma exchanges. Three single exchanges did not improve her status. A series of intensive plasma exchanges of 6 liters each on 4 consecutive days reduced her anti-acetylcholine receptor antibodies to one-tenth of the initial value. The reduction was accompanied by an impressive clinical improvement. This study confirms that antibodies can be removed by plasma exchange. It further shows that the clinical improvement appears to be closely related to the quantity of antibodies removed.

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.

An SH2-domain-containing kinase negatively regulates the phosphatidylinositol-3 kinase pathway.

SHK1 is a novel dual-specificity kinase that contains an SH2 domain in its C-terminal region. We demonstrate that SHK1 is required for proper chemotaxis and phagocytosis. Mutant shk1 null cells lack polarity, move very slowly, and exhibit an elevated and temporally extended chemoattractant-mediated activation of the kinase Akt/PKB. GFP fusions of the PH domain of Akt/PKB or the PH-domain-containing protein CRAC, which become transiently associated with the plasma membrane after a global stimulation with a chemoattractant, remain associated with the plasma membrane for an extended period of time in shk1 null cells. These results suggest that SHK1 is a negative regulator of the PI3K (phosphatidylinositol-3 kinase) pathway. Furthermore, when a chemoattractant gradient is applied to a wild-type cell, these PH-domain-containing proteins and the F-actin-binding protein coronin localize to its leading edge, but in an shk1 null cell they become randomly associated with the plasma membrane and cortex, irrespective of the direction of the chemoattractant gradient, suggesting that SHK1 is required for the proper spatiotemporal control of F-actin levels in chemotaxing cells. Consistent with such functions, SHK1 is localized at the plasma membrane/cortex, and we show that its SH2 domain is required for this localization and the proper function of SHK1.