Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen.

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.     

The tumor suppressor p53 is bound to RNA by a stable covalent linkage.

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.     

The phosphorylation at Thr 124 of simian virus 40 large T antigen is crucial for its oligomerization

SV40 large T antigen is phosphorylated at up to ten different amino acids clustered in an N-terminal and a C-terminal part of the polypeptide chain. The N-terminal phosphorylated residues include Ser 123 and Thr 124. We have analyzed the oligomerization, the complex formation with the cellular oncoprotein p53 and the DNA-binding properties of T antigen from two different SV40 transformed cell lines which have either an amino acid exchange at Ser 123 to Phe (W7) or Thr 124 to Ile (D29). In comparison to wild-type T antigen both mutant T antigens have a slightly reduced binding affinity for both binding sites, I and II, of SV40 DNA. Phosphorylation at both residues of T antigen is not essential for formation of the complex with p53. Only the phosphorylation at Thr 124 seems to be critical for the formation of high molecular mass oligomers. Our data support the hypothesis that the oligomerization of T antigen seems to be implicated in viral DNA replication.     

Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography

Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using liquid chromatography-electrospray ionization tandem mass spectrometry, 14 phosphorylation sites were identified on Xenopus laevis His6-Aurora A, a highly conserved regulator of centrosome maturation and cell division. These included seven novel phosphorylation sites, Ser-12, Thr-21, Thr-103, Ser-116, Thr-122, Tyr-155, and Thr-294, as well as the previously identified regulatory sites, Ser-53, Thr-295, and Ser-349. The identification of these novel phosphorylation sites will be important for future studies aimed at elucidating the mechanisms of Aurora A regulation by phosphorylation. Furthermore, we demonstrate that a "kinase-inactive" mutant of Aurora A, K169R, still retains 10% of activity of the wild-type enzyme in vitro along with occupancy of Thr-295 and Ser-12. However, mutation of Asp-281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Because of the abundance of phosphorylated residues on His6-Aurora A, we found this protein to be an ideal tool for the characterization of immobilized metal-affinity chromatography (IMAC) as a method for phosphopeptide enrichment from complex mixtures. We present a detailed analysis of the binding and elution properties of both the phosphopeptides and unphosphorylated peptides of His6-Aurora A to Fe3+-IMAC before and after methyl esterification. Moreover, we demonstrate a significant difference in enrichment of phosphopeptides when different resins are used for Fe3+-IMAC and characterize the strengths and limitations of this methodology for the study of phosphoproteomics.     

The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen.

We have compared the ATPase, DNA-binding, and helicase activities of free simian virus 40 (SV40) large T antigen (To) and T antigen complexed with cellular p53 (T+p53). Each activity is essential for productive viral infection. The T+p53 and To fractions were prepared by sequential immunosorption of infected monkey cells with monoclonal antibodies specific for p53 and T antigen. The immune-complexed T fractions were then assayed in parallel. For ATP hydrolysis, the Vmax for T+p53 was 143 nmol of ADP per min per mg of protein, or 18-fold greater than for To. ATP had no effect on the stability of the T+p53 complex. The T+p53 complex was significantly more active than To in hydrolyzing dATP, dGTP, GTP, and UTP. Of the nucleotide substrates tested, the greatest relative increase (T+p53/To) was in hydrolyzing dGTP and GTP. In DNase footprinting assays performed under replication conditions, the T+p53 complex protected regions I, II, and III of origin DNA while equivalent amounts of To protected only regions I and II. Region III is known to contribute to the efficiency of DNA replication and contains the SP1-binding sites of the early viral promoter. The T+p53 fraction was also a more efficient helicase than To, especially with a GC-rich primer and template. Thus, the T+p53 complex has enhanced ATPase, GTPase, DNA-binding, and helicase activities. These findings imply that complex formation between cellular monkey p53 and SV40 T antigen modulates a number of essential activities of T in SV40 productive infection.     

Novel protein phosphorylation site identification in spinach stroma membranes by titanium dioxide microcolumns and tandem mass spectrometry

In this work, spinach stroma membrane, instead of thylakoid, has been investigated for the presence of phosphorylated proteins. We identified seven previously unknown phosphorylation sites by taking advantage of TiO(2) phosphopeptides enrichment coupled to mass spectrometric analysis. Upon illumination at 100 micromol m(-2) s(-1), two novel phosphopeptides belonging to the N-terminal region of Lhcb1 light-harvesting protein were detected: NVSSGS(p)PWYGPDR and T(p)VQSSSPWYGPDR. Moreover, three new threonine residues in CP43 (Thr-6, Thr-8, and Thr-346) and, for the first time, two amino acid residues of the N-terminus of Rieske Fe-S protein of the cytochrome b(6)f complex (Thr-2 and Ser-3) were revealed to be phosphorylated. Since Lhcb1 and CP43 have been reported as mobile proteins, it may be suggested that illumination derived phosphorylation, and consequently the addition of negatively charged groups to the protein, is a necessary condition to induce a significant protein structural change.     

Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells.

We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.     

Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40.

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been analyzed by partial proteolysis peptide mapping and phosphoamino acid analysis of the resulting products. At least four sites were found to be phosphorylated. An amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the proline-rich carboxy-terminal end of the molecule, either at position 701 or at position 708. The mutant dl 1265, which is defective in adenovirus helper function, lacked this phosphorylation site. In addition, the carboxy-terminal part of the molecule contained phosphoserine at a more central position. T-antigen-associated proteins of SV40-transformed cell (nonviral T; 51,000 to 55,000 daltons) also contained multiple phosphorylation sites involving at least two serine residues in mouse antigens and an additional threonine residue in rat, human, and monkey antigens. The latter residue and at least one phosphoserine residue were located near one terminus of the human NVT molecule. We did not find any evidence for phosphorylation of tyrosine residues in any of the multiple species of either large T or nonviral T molecules. Several forms of large T antigens were extracted from both SV40-transformed and SV40-infected permissive and nonpermissive cells, and their phosphorylation patterns were compared. No evidence was found for a different phosphorylation pattern of T antigen in transformed cells.     

The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.     

Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.     

Two populations of p27 use differential kinetics to phosphorylate Ser-10 and Thr-187 via phosphatidylinositol 3-Kinase in response to fibroblast growth factor-2 stimulation

The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G(1)/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G(1)/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G(1) phase of the cell cycle.     

Stable T-p53 complexes are not required for replication of simian virus 40 in culture or for enhanced phosphorylation of T antigen and p53.

We generated a number of simian virus 40 (SV40) mutants with single amino acid substitutions in T antigen between residues 388 and 411. All but one mutant (398LV) replicated like wild-type SV40 and gave rise to normal-size plaques. Three different mutations at residue 402 (Asp to Glu, Asn, or His) totally prevented the formation of stable complexes with the cellular protein p53 in monkey cells but had no effect on virus replication. Only one other mutation in this region, involving residue 401 (Met to Thr), slightly inhibited the formation of T-monkey p53 complexes. The three mutant T antigens with substitutions at residue 402 also formed no stable complexes with human p53 but generated low levels of complexes with mouse p53. These results indicate that residue 402 is critical for binding to monkey and human p53 proteins and is important for binding to mouse p53. We suggest that it is one of several points of contact. In cells infected with any one of the three residue 402 mutant viruses. T antigen and p53 became increasingly phosphorylated, as they were in cells infected with wild-type virus. Our data therefore show that stable T-p53 complexes are not required for replication of SV40 in culture or for enhanced phosphorylation of either protein.     

MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection.

Phosphopeptide analyses of the simian virus 40 (SV40) large tumor antigen (LT) in SV40-transformed rat cells, as well as in SV40 lytically infected monkey cells, showed that gel-purified LT that was not complexed to p53 (free LT) and p53-complexed LT differed substantially in their phosphorylation patterns. Most significantly, p53-complexed LT contained phosphopeptides not found in free LT. We show that these additional phosphopeptides were derived from MDM2, a cellular antagonist of p53, which coprecipitated with the p53-LT complexes, probably in a trimeric LT-p53-MDM2 complex. MDM2 also quantitatively bound the free p53 in SV40-transformed cells. Free LT, in contrast, was not found in complex with MDM2, indicating a specific targeting of the MDM2 protein by SV40. This specificity is underscored by significantly different phosphorylation patterns of the MDM2 proteins in normal and SV40-transformed cells. Furthermore, the MDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of MDM2 by SV40 is aimed at preventing MDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells.     

Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.     

In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells

Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis.     

N-terminal Serine Dephosphorylation Is Required for KCC3 Cotransporter Full Activation by Cell Swelling

The K⁺:Cl⁻ cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.     

Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215

The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 posttranslational modifications and association with other proteins have been implicated in the regulation of its stability and transactivation activity. Here we show that p53 is phosphorylated by the mitotic kinase Aurora-A at serine 215. Unlike most identified phosphorylation sites of p53 that positively associate with p53 function (Brooks, C. L., and Gu, W. (2003) Curr. Opin. Cell Biol. 15, 164-171), the phosphorylation of p53 by Aurora-A at Ser-215 abrogates p53 DNA binding and transactivation activity. Downstream target genes of p53, such as p21Cip/WAF1 and PTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e. phosphomimic p53-S215D lost and non-phosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and gamma-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53 (Katayama, H., Sasai, K., Kawai, H., Yuan, Z. M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R. B., Czerniak, B. A., and Sen, S. (2004) Nat. Genet. 36, 55-62). Our data indicate that phosphorylation of p53 at Ser-215 by Aurora-A is a major mechanism to inactivate p53 and can provide a molecular insight for Aurora-A function.     

Phosphorylation of the membrane proximal region of tumor necrosis factor receptor CD120a (p55) at ERK consensus sites

The interaction of tumor necrosis factor-alpha with its receptor CD120a (p55) initiates downstream signaling cascades that include the activation of the mitogen-activated protein kinase (MAPK), p42(mapk/erk2). The membrane proximal region of CD120a (p55) is Ser-, Thr-, and Pro-rich and contains four mitogen-activated protein kinase consensus phosphorylation sites. In recent work, we showed that CD120a (p55) itself is a target of phosphorylation by p42(mapk/erk2), and after phosphorylation, the receptor is redistributed from the cell surface and Golgi complex to intracellular tubular structures associated with elements of the endoplasmic reticulum. The goal of this study was to define the specific amino acid residues that are phosphorylated. Deletional mutagenesis of the cytoplasmic domain of CD120a (p55) indicated that two sites located between residues 207-254 and 250-300 were phosphorylated predominantly on Thr and Ser residues, respectively. Site-directed mutagenesis of Ser and Thr residues contained within the extracellular signal-regulated kinase (ERK) consensus sequences indicated that the preferred residues were Thr-236 and Ser-270. Primary phosphorylation at these sites appeared to enable subsequent phosphorylation at Ser-240 and Ser-244, although the level of phosphorylation of these latter two sites was less than the preferred sites. Through the use of specific ligation of CD120a (p55) alone and mice deficient in CD120a (p55), CD120b (p75), or both receptors, CD120a (p55) was shown to be necessary and sufficient for the induction of kinase activity. These findings thus suggest that the phosphorylation of Thr-236 and Ser-270 within the membrane proximal region of CD120a (p55) are the preferred sites of phosphorylation by p42(mapk/erk2) and may set in motion phosphorylation at other sites.     

Identification of in vivo phosphorylation sites and their functional significance in the sodium iodide symporter

The Na+/I- symporter (NIS)-mediated iodide uptake activity is the basis for targeted radioiodide ablation of thyroid cancers. Although it has been shown that NIS protein is phosphorylated, neither the in vivo phosphorylation sites nor their functional significance has been reported. In this study, Ser-43, Thr-49, Ser-227, Thr-577, and Ser-581 were identified as in vivo NIS phosphorylation sites by mass spectrometry. Kinetic analysis of NIS mutants of the corresponding phosphorylated amino acid residue indicated that the velocity of iodide transport of NIS is modulated by the phosphorylation status of Ser-43 and Ser-581. We also found that the phosphorylation status of Thr-577 may be important for NIS protein stability and that the phosphorylation status of Ser-227 is functionally silent. Thr-49 appears to be critical for proper local structure/conformation of NIS because mutation of Thr-49 to alanine, aspartic acid, or serine results in reduced NIS activity without alterations in total or cell surface NIS protein levels. Taken together, we showed that NIS protein levels and functional activity could be modulated by phosphorylation through distinct mechanisms.     

Involvement of aberrant glycosylation in phosphorylation of tau by cdk5 and GSK-3beta

Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in the brains of individuals with Alzheimer’s disease (AD). We recently found that the glycosylation might precede hyperphosphorylation of tau in AD. In this study, we investigated the effect of glycosylation on phosphorylation of tau catalyzed by cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase-3β (GSK-3β). The phosphorylation of the longest isoform of recombinant human brain tau, tau₄₄₁, at various sites was detected by Western blots and by radioimmuno-dot-blot assay with phosphorylation-dependent and site-specific tau antibodies. We found that cdk5 phosphorylated tau₄₄₁ at Thr-181, Ser-199, Ser-202, Thr-205, Thr-212, Ser-214, Thr-217, Thr-231, Ser-235, Ser-396, and Ser-404, but not at Ser-262, Ser-400, Thr-403, Ser-409, Ser-413, or Ser-422. GSK-3β phosphorylated all the cdk5-catalyzed sites above except Ser-235. Deglycosylation by glycosidases depressed the subsequent phosphorylation of AD-tau (i) with cdk5 at Thr-181, Ser-199, Ser-202, Thr-205, and Ser-404, but not at Thr-212; and (ii) with GSK-3β at Thr-181, Ser-202, Thr-205, Ser-217, and Ser-404, but not at Ser-199, Thr-212, Thr-231, or Ser-396. These data suggest that aberrant glycosylation of tau in AD might be involved in neurofibrillary degeneration by promoting abnormal hyperphosphorylation by cdk5 and GSK-3β.