Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein

Regulatory elements interacting with DNA-binding proteins have been investigated in the promoter sequence of the early PE38 gene in the Autographa californica nuclear polyhedrosis virus (AcNPV). A GATA motif located 50 nucleotides upstream of the PE38 transcriptional start site is recognized differentially in the course of infection. As demonstrated by footprint and gel mobility shift assays, the GATA sequences TTATCT are protected by nuclear extracts from uninfected Spodoptera frugiperda cells and from S. frugiperda cells early postinfection (p.i.) but not by S. frugiperda cell extracts isolated 40 h p.i. We have compared the binding capacity of the insect GATA-like protein with that of the vertebrate GATA-1 factor identified as erythroid-specific factor. Our results indicate that a factor present in mouse erythroleukemia cells, presumably GATA-1, can bind to the insect GATA motif and vice versa. Evidence from transient expression studies suggests that the mutated GATA sequences do not influence PE38 promoter activity in cell culture.</jats:p

Characterization of Regulatory Events Associated with Membrane Targeting of p90 Ribosomal S6 Kinase 1

RSK is a serine/threonine kinase containing two distinct catalytic domains. Found at the terminus of the Ras/extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) kinase cascade, mitogen-stimulated ribosomal S6 kinase (RSK) activity requires multiple inputs. These inputs include phosphorylation of the C-terminal kinase domain activation loop by ERK1/2 and phosphorylation of the N-terminal kinase domain activation loop by phosphoinositide-dependent protein kinase-1 (PDK1). Previous work has shown that upon mitogen stimulation, RSK accumulates in the nucleus. Here we show that prior to nuclear translocation, epidermal growth factor-stimulated RSK1 transiently associates with the plasma membrane. Myristylation of wild-type RSK1 results in an activated enzyme in the absence of added growth factors. When RSK is truncated at the C terminus, the characterized ERK docking is removed and RSK phosphotransferase activity is completely abolished. When myristylated, however, this myristylated C-terminal truncated form (myrCTT) is activated at a level equivalent to myristylated wild-type (myrWT) RSK. Both myrWT RSK and myrCTT RSK can signal to the RSK substrate c-Fos in the absence of mitogen activation. Unlike myrWT RSK, myrCTT RSK is not further activated by serum. Only the myristylated RSK proteins are basally phosphorylated on avian RSK1 serine 381, a site critical for RSK activity. The myristylated and unmyristylated RSK constructs interact with PDK1 upon mitogen stimulation, and this interaction is insensitive to the MEK inhibitor UO126. Because a kinase-inactive CTT RSK can be constitutively activated by targeting to the membrane, we propose that ERK may have a dual role in early RSK activation events: preliminary phosphorylation of RSK and escorting RSK to a membrane-associated complex, where additional MEK/ERK-independent activating inputs are encountered

VEGF: A modifier of the del22q11 (DiGeorge) syndrome?

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.status: publishe

Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling

Protein kinase C (PKC) is involved in a wide array of cellular processes such as cell proliferation, differentiation and apoptosis. Phosphorylation of both turn motif (TM) and hydrophobic motif (HM) are important for PKC function. Here, we show that the mammalian target of rapamycin complex 2 (mTORC2) has an important function in phosphorylation of both TM and HM in all conventional PKCs, novel PKCɛ as well as Akt. Ablation of mTORC2 components (Rictor, Sin1 or mTOR) abolished phosphorylation on the TM of both PKCα and Akt and HM of Akt and decreased HM phosphorylation of PKCα. Interestingly, the mTORC2-dependent TM phosphorylation is essential for PKCα maturation, stability and signalling. Our study demonstrates that mTORC2 is involved in post-translational processing of PKC by facilitating TM and HM phosphorylation and reveals a novel function of mTORC2 in cellular regulation

Hydrophobic Motif Phosphorylation Coordinates Activity and Polar Localization of the Neurospora crassa Nuclear Dbf2-Related Kinase COT1

Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as a conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2