DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro.

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

Regulation of peroxisome proliferator-activated receptor alpha by protein kinase C

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acids, hypolipidemic drugs, and peroxisome proliferators (PPs). Like other nuclear receptors, PPARalpha is a phosphoprotein whose activity is affected by a variety of growth factor signaling cascades. In this study, the effects of protein kinase C (PKC) on PPARalpha activity were explored. In vivo phosphorylation studies in COS-1 cells transfected with murine PPARalpha showed that the level of phosphorylated PPARalpha is increased by treatment with the PP Wy-14,643 as well as the PKC activator phorbol myristol acetate (PMA). In addition, inhibitors of PKC decreased Wy-14,643-induced PPARalpha activity in a variety of reporter assays. Overexpressing PKCalpha, -beta, -delta, and -zeta affected both basal and Wy-14,643-induced PPARalpha activity. Four consensus PKC phosphorylation sites are contained within the DNA binding (C-domain) and hinge (D-domain) regions of rat PPARalpha (S110, T129, S142, and S179), and their contribution to receptor function was examined. Mutation of T129 or S179 to alanine prevented heterodimerization of PPARalpha with RXRalpha, lowered the level of phosphorylation by PKCalpha and PKCdelta in vitro, and lowered the level of phosphorylation of transfected PPARalpha in transfected cells. In addition, the T129A mutation prevented PPARalpha from binding DNA in an electromobility shift assay. Together, these studies demonstrate a direct role for PKC in the regulation of PPARalpha, and suggest several PKCs can regulate PPARalpha activity through multiple phosphorylation sites.     

Phosphorylation and/or Presence of Serine 37 in the Movement Protein of Tomato Mosaic Tobamovirus Is Essential for Intracellular Localization and Stability In Vivo

The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serine 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the subcellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of Aequorea victoria. Wild-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable than wild-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function.     

Phosphorylation of StarD10 on serine 284 by casein kinase II modulates its lipid transfer activity

StarD10 is a dual specificity lipid transfer protein capable of shuttling phosphatidylcholine and phosphatidylethanolamine between membranes in vitro. We now provide evidence that, in vivo, StarD10 is phosphorylated on serine 284. This novel phosphorylation site was identified by tandem mass spectrometry of immunoaffinity-purified StarD10 from lysates of HEK293T cells transiently expressing the protein. In vitro kinase assays revealed that casein kinase II was capable of phosphorylating wild-type StarD10 but not a S284A mutant protein. Interestingly, hypotonic extracts prepared from HEK293T cells expressing the serine to alanine mutant exhibited increased lipid transfer activity compared with those from wild-type StarD10-expressing cells, suggesting that, in a cellular context, phosphorylation on serine 284 negatively regulates StarD10 activity. Because casein kinase II phosphorylation also inhibited lipid transfer activity of the purified recombinant StarD10 protein, inhibition is not dependent on any cellular cofactors. Instead, our data show that C-terminal StarD10 phosphorylation on serine 284 regulates its association with cellular membranes.     

Protein kinase C-dependent enhancement of activity of rat brain NCKX2 heterologously expressed in HEK293 cells

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.     

Investigations of the potential effects of phosphorylation of the MWFE and ESSS subunits on complex I activity and assembly

There have been several reports on the phosphorylation of various subunits of NADH-ubiquinone oxidoreductase (complex I) in mammalian mitochondria. The effects of phosphorylation on assembly or activity of these subunits have not been investigated directly. The cAMP-dependent phosphorylation of the MWFE and ESSS subunits in isolated bovine heart mitochondria has been recently reported. We have investigated the significance of potential phosphorylation of these two subunits in complex I assembly and function by mutational analysis of the phosphorylation sites. Chinese hamster mutant cell lines missing either the MWFE or the ESSS subunits were transfected and complemented with the corresponding wild type and mutant cDNAs made by site-directed mutagenesis. In MWFE the serine 55 was substituted by alanine, glutamate, glutamine, and aspartate (S55A, S55E, S55Q, and S55D, respectively). The glutamate substitutions might be expected to mimic the phosphorylated state of the protein. With the exception of the MWFE(S55A) mutant protein the assembly of complex I was completely blocked, and no activity could be detected. Various substitutions in the ESSS protein (S2A, S2E, S8A, S8E, T21A, T21E, S30A, S30E) appeared to cause lower levels of mature protein and a significantly reduced complex I activity measured polarographically. The ESSS (S2/8A) double mutant protein caused a complete failure to assemble. These mutational analyses suggest that if phosphorylation occurs in vivo, the effects on complex I activity are significant.     

Differential regulation of phosphoglucose isomerase/autocrine motility factor activities by protein kinase CK2 phosphorylation

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser(185) by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser(185) with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. The three recombinant mutant proteins exhibited indistinguishable cytokine activity and receptor-binding affinities compared with the wild-type protein. In both in vitro and in vivo assays, the wild-type and S185A mutant proteins underwent active species dimerization, whereas both the S185D and S185E mutant proteins also formed tetramers. These results demonstrate that phosphorylation affects the allosteric kinetic properties of the enzyme, resulting in a less active form of PGI, whereas non-phosphorylated protein species retain cytokine activity. The process by which phosphorylation modulates the enzymatic activity of PGI thus has an important implication for the understanding of the biological regulation of this key glucose metabolism-regulating enzyme.     

Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.     

Asymmetric processing of human immunodeficiency virus type 1 cDNA in vivo: implications for functional end coupling during the chemical steps of DNA transposition

Retroviral integration, like all forms of DNA transposition, proceeds through a series of DNA cutting and joining reactions. During transposition, the 3' ends of linear transposon or donor DNA are joined to the 5' phosphates of a double-stranded cut in target DNA. Single-end transposition must be avoided in vivo because such aberrant DNA products would be unstable and the transposon would therefore risk being lost from the cell. To avoid suicidal single-end integration, transposons link the activity of their transposase protein to the combined functionalities of both donor DNA ends. Although previous work suggested that this critical coupling between transposase activity and DNA ends occurred before the initial hydrolysis step of retroviral integration, work in the related Tn10 and V(D)J recombination systems had shown that end coupling regulated transposase activity after the initial hydrolysis step of DNA transposition. Here, we show that integrase efficiently hydrolyzed just the wild-type end of two different single-end mutants of human immunodeficiency virus type 1 in vivo, which, in contrast to previous results, proves that two functional DNA ends are not required to activate integrase's initial hydrolysis activity. Furthermore, despite containing bound protein at their processed DNA ends, these mutant viruses did not efficiently integrate their singly cleaved wild-type end into target DNA in vitro. By comparing our results to those of related DNA recombination systems, we propose the universal model that end coupling regulates transposase activity after the first chemical step of DNA transposition.     

Epigenetic effect of the rad201(G1) mutation in a system with mobilization of nonautonomous P elements in Drosophila

The effect on P-element activity in somatic cells was studied for a repair mutation localized in the Drosophila genome in the region of the rad201 and Rad51C overlapping genes. When one of the parents carried nonautonomous P elements and the rad201 mutation and the other carried a P-transposase source, a high dominant pupal lethality was observed in the progeny heterozygous for the mutant allele and their sibs homozygous for the rad201+ wild-type allele. The sib response was due to the epigenetic effect of the rad201 mutation and was maintained through at least two generations. The specifics of the epigenetic effect are discussed in terms of its possible association with P transpositions and mitotic crossing over events determined by P transposase. Based on the results of genetic and genomic DNA analyses of the rad201 mutant, it was assumed that the phenomenon in question was determined by several genetic factors.     

Replication protein A (RPA) phosphorylation prevents RPA association with replication centers

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2(D)) or alanine (RPA2(A)). Although RPA2(D) was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2(D) mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2(D) again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2(D). In contrast, under DNA damage or replication stress conditions, RPA2(D), like RPA2(A) and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with gamma-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.     

Dual requirement for a newly identified phosphorylation site in p70s6k.

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.     

Characterization of two hypertransposing Tn5 mutants.

Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh). Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345). The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans. The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays. During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins. We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting. The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species.     

Herpes simplex virus 1 protein kinase Us3 and major tegument protein UL47 reciprocally regulate their subcellular localization in infected cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells.