Lack Of Phosphorylation Research Articles

MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis

The myocyte enhancer factor 2B (MEF2B) transcription factor is frequently mutated in germinal center (GC)-derived B-cell lymphomas. Its ammino (N)-terminal mutations drive lymphomagenesis by escaping interaction with transcriptional repressors, while the function of carboxy (C)-terminal mutations remains to be elucidated. Here, we show that MEF2B C-tail is physiologically phosphorylated at specific residues and phosphorylation at serine (S)324 is impaired by lymphoma-associated mutations. Lack of phosphorylation at S324 enhances the interaction of MEF2B with the SWI/SNF chromatin remodeling complex, leading to higher transcriptional activity. In addition, these mutants show an increased protein stability due to impaired interaction with the CUL3/KLHL12 ubiquitin complex. Mice expressing a phosphorylation-deficient lymphoma-associated MEF2B mutant display GC enlargement and develop GC-derived lymphomas, when crossed with Bcl2 transgenic mice. These results unveil converging mechanisms of action for a diverse spectrum of MEF2B mutations, all leading to its dysregulation and GC B-cell lymphomagenesis.

Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53Ser46 mutant.

Genetic mutations in p53 are frequently associated with many types of cancers that affect its stability and activity through multiple ways. The Ser46 residue present in the transactivation domain2 (TAD2) domain of p53 undergoes phosphorylation that blocks its degradation by MDM2 and leads to cell cycle arrest/apoptosis/necrosis upon intrinsic or extrinsic stresses. On the other hand, unphosphorylated p53 mutants escape cell arrest or death triggered by these molecular signaling axes and lead to carcinogenesis. Phosphorylation of Ser in the TAD2 domain of p53 mediates its interactions with transcription factor p62, yielding transcriptional activation of downstream pro-apoptotic genes. The p53 phosphorylation causes string-like elongated conformation that increases its binding affinity with the PH domain of p62. On the other hand, lack of phosphorylation causes helix-like motifs and low binding affinity to p62. We undertook molecular simulation analyses to investigate the potential of some natural small molecules (Withanone (Wi-N) & Withaferin-A (Wi-A) from Ashwagandha; Cucurbitacin-B (Cuc-B) from bitter Cucumber; and Caffeic acid phenethyl ester (CAPE) and Artepillin C (ARC) from honeybee propolis) to interact with p62-binding region of p53 and restore its wild-type activity. We found that Wi-N, Wi-A, and Cuc-B have the potential to restore p53-p62 interaction for phosphorylation-deficient p53 mutants. Wi-N, in particular, caused a reversal of the α-helical structure into an elongated string-like conformation similar to the wild-type p53. These data suggested the use of these natural compounds for the treatment of p53Ser46 mutant harbouring cancers. We also compared the efficiency of Wi-N, Wi-A, Cuc-B, CAPE, and ARC to abrogate Mortalin-p53 binding resulting in nuclear translocation and reactivation of p53 function and provide experimental evidence to the computational analysis. Taken together, the use of these small molecules for reactivation of p53 in cancer cells is suggested.

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.

RB Regulates DNA Double Strand Break Repair Pathway Choice by Mediating CtIP Dependent End Resection.

Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.

Fibrinogen Activates FAK to Promote the Colorectal Adenocarcinoma Growth

Along with other proteins in the coagulation cascade, including tissue factor and thrombin, fibrinogen has been shown to promote tumor metastasis across numerous cancer types. However, the role of fibrin(ogen) in primary tumor growth is context dependent and not universally important. One cancer strongly dependent on fibrin(ogen) for primary tumor growth is colorectal cancer (CRC), but the mechanisms by which fibrinogen supports colon cancer growth are not well understood. To delineate the mechanism of fibrin(ogen)-supported tumor growth, we implanted C57BL/6-derived murine colon cancer cells (MC38) into the dorsal subcutis of syngeneic mice with specific deletions/alteration in fibrinogen or FXIII-deficiency. Complete fibrinogen deficiency significantly limited the growth of colon cancer cells. However, the imposition of FXIII-deficiency or specific mutations in fibrinogen that 1) prevent the formation of fibrin polymer (FibAEK), limit fibrin(ogen) binding to the integrin αMβ2 (Fibɣ390-396A), or remove the ɣ chain binding motif for the platelet integrin αIIbβ3 (FibɣΔ5) had no impact on colon cancer growth. To explore the mechanisms coupling fibrin(ogen) to colon cancer growth using a nonbiased approach, we performed RNA-Seq analyses of murine CRC tumors harvested from Fib+ and Fib- mice. We detected significant differences in the expression of 214 genes (128 downregulated and 86 upregulated) involved in cellular proliferation, survival, migration and metabolism. Notably, Stratifin (SFN), which encodes 14-3-3-σ, was one of the genes found to be highly upregulated in tumors from Fib- mice relative to Fib+ mice. 14-3-3-σ is a potent cell cycle regulator and it is also known to stabilize p53, which ultimately inhibits tumor growth. In a separate validation cohort, we observed significantly increased protein expression of 14-3-3-σ and its upstream and downstream targets (i.e., p53, p21 and p27) in tumors harvested from Fib- mice relative to controls. We also compared FAK activation, a key negative regulator of p53, in tumors from Fib+ and Fib- mice. FAK was inactive in tumors from Fib- mice, as indicated by lack of phosphorylation at tyrosine 397 (p-FAK Tyr397). Also, MDM2 was less phosphorylated at Ser166 in Fib- tumors, suggesting that p53 is not degraded by MDM2 in the absence of fibrinogen. Taken together, these data suggest that fibrin(ogen)-mediated downregulation of p53 and other targets via FAK activation and downregulation of 14-3-3-σ results in senescence of CRC cells. Consistent with this view, Ki67 positive nuclei were significantly less in tumor from Fib- mice relative to controls We also observed senescence-associated-β-galactosidase (SA-β-gal) activity in the tumors from Fib- mice, but not those from Fib+ animals. Furthermore, NMR-based metabolomics analyses demonstrated significantly less NAD+ and lactate levels in tumors from Fib- mice, which further confirms that fibrinogen deficiency hampers proliferation by inhibiting major metabolic pathways. In order to determine if fibrinogen-mediated support of CRC growth is tumor cell intrinsic, we compared MC38 growth in the 3D bioprinted matrices consisting of fibrin or albumin. MC38 cells showed a higher proliferation rate in the fibrinogen printed 3D environment compared to the albumin environment. These findings suggest that fibrin(ogen)-mediated engements of tumor cells activates FAK which inhibits p53 and its downstream targets including 14-3-3-σ and p21 which promote cellular proliferation and prevent senescence. Overall, these studies suggest that fibrin(ogen) is the important component of the CRC microenvironment and could be exploited for targeting and treating the CRC. Disclosures Whitlock: POSNA: Other: Research Committee; MTF Biologics Grant Program: Other: site co-investigator. Palumbo:Ionis Pharmaceuticals: Research Funding.

Abstract 5447: Overexpression of DARPP-32 promotes activation of STAT3 through IGF1R-SRC axis in gastric cancer

Abstract Background: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is a novel cancer gene overexpressed in 2/3 of gastric cancer patients. DARPP-32 promotes cancer cell survival, drug resistance, and invasion. Activation of STAT3, signal transducer and activator of transcription 3, is important for tumorigenesis and drug resistance. Among factors that regulate STAT3, SOCS3 acts as a negative regulator of STAT3 to prevent its uncontrolled oncogenic activation. In this study, we aimed to investigate if DARPP-32 regulates STAT3 and determine the underlying molecular mechanisms. Methods: The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. The association between DARPP-32 and STAT3 was evaluated by immunofluorescence. SOCS3 degradation was analyzed by Ubiquitination Assay. The interaction between DARPP-32 and IGF1R was evaluated by co-immunoprecipitation assays. Luciferase reporter and immunohistochemistry stain were used in this study. Results: Overexpression of DARPP-32 in AGS gastric cancer cells increased phosphorylation of STAT3 (Y705) and activation of STAT3 luciferase reporter (P<0.01), and decreased SOCS3 protein expression. DARPP-32 expression increased phosphorylation and ubiquitination of the SOCS3 protein, and subsequently shortened the SOCS3 protein half-life. We provide evidence showing that DARPP-32 promotes activation of IGF1R-SRC axis, critical for phosphorylation and degradation of SOCS3, a negative regulator of STAT3 activity. Proximity ligation and co-immunoprecipitation assays indicated that DARPP-32 and IGF1R co-exist in the same protein complex where DARPP-32 binding to IGF1R promotes its phosphorylation with activation of downstream SRC and STAT3. We obtained double knockouts mice (Darpp32KO/Tff1KO) to determine the histological changes at 3 age groups (3, 6, 9- 12 month). Using the different age groups, we found that the double knockout mouse model have significantly lower incidence of LGD at the age of 3 month, as compared to TFF1 KO (P<0.05) mice. The Darpp32KO/Tff1KO gastric mucosa demonstrated lack of phosphorylation and nuclear localization of STAT3 with significant delay in the onset of tumor lesions. Immunohistochemistry analysis confirmed overexpression of DARPP-32 and nuclear localization of STAT3 in human gastric cancers. Conclusion: The in vitro studies indicate that DARPP-32 plays a critical role in activation of STAT3 signaling through regulation of IGF1R and SRC, leading to SOCS3 degradation. In vivo results suggest that lack of DARPP-32 delays the development of tumors in the TFF1 knockout mouse model but is not sufficient alone to abrogate the full carcinogenesis cascade. These novel findings add to the oncogenic functions of DARPP-32 and highlight its potential role in the pro-inflammatory gastric carcinogenesis cascade. Citation Format: Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai. Overexpression of DARPP-32 promotes activation of STAT3 through IGF1R-SRC axis in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5447.

Abstract B72: Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line

Abstract The deregulation of p90 ribosomal S6 kinases (RSKs) seems to be responsible for different oncogenic outcomes in several types of tumors. The effects on mRNA translation regulation via RSKs in glioblastomas (GBMs) were not described so far; thus it is essential to understand the molecular pathways regulated by RSKs that control protein synthesis in GBM. Recent data from our group (unpublished) indicate that different GBM cell lines express distinct levels of the RSK1 isoform, while RSK2 levels are much less variable, and RSK3 and RSK4 are not detected. LN-18 cells show higher levels of RSK1 when compared to A172 or U-118 MG; thus CRISPR/Cas9 system technology was used in this cell line to generate RSK-deficient cells. Briefly, two different sets of small guide RNAs directed to both RSK1 and RSK2 were constructed. LN-18 cells were transfected to express Cas9 along with either one of the constructs targeting RSK1 and/or RSK2. Selection of individual cell clones was followed by Western blotting detection of RSK1 and RSK2 isoforms to identify RSK-deficient cells. We were able to efficiently generate clones with no detection of RSK1 (RSK1-/-), RSK2 (RSK2-/-) with both sets of sgRNAs and cells that had both isoforms knocked out (RSK1-/-/RSK2-/-) only with one of the sgRNA sets. Considering that mTORC1 signaling pathway is the main mediator of RSKs-dependent translation activation, the crosstalk between these pathways and its effects on translation levels were studied in RSKs-deficient cells. Therefore, cells were serum starved for 48 h and stimulated with phorbol-12-myristate-13-acetate (PMA) to activate the Ras-ERK1/2-RSK pathway. Signaling pathways involved in RSK and mTORC1 translation activation were assessed. Interestingly, RSK-deficient cells show lower levels of phosphorylation of Tuberous Sclerosis Complex 2 (TSC-2) at 1798 serine residue, confirming the importance of RSK phosphorylation on this residue and its relevance for evaluation of active RSK levels on cells. In order to describe the influence of RSK1 in the translation initiation rate of GBM cells, polysome profiling was assayed in LN-18, A172, and U-118 MG cells, containing high, low, or nondetected levels of RSK1, respectively. Treatment with PMA for 1, 2, or 4 hours affected translation in GBM cells in a different manner, with the cell lines that display lower levels of RSK1 being less sensitive to the treatment, when compared to LN-18 cells. Polysome profiling was also performed with CRISPR/Cas9-generated cells. RSK1-/- and RSK2-/- cells showed reduced translation initiation levels in basal conditions; however, translation initiation was stimulated after PMA treatment, probably because the remaining isoform is enough to elicit this response. Surprisingly, translation levels were comparable between control cells and RSK1-/-/RSK2-/- cells, suggesting the presence of a compensatory mechanism during the generation of double knockout cells. Of note, this same response could be observed in cells that display alterations in the elongation phase, as it can occur by the lack of phosphorylation of eEF2 kinase in the absence of RSKs. In conclusion, the generation of RSKs-deficient GBM-cell line by the CRISPR/Cas9 system is a useful model for studying and characterizing RSK effects on signaling and translation in GBM cells. Supported by FAPESP. Citation Format: Danielle Nascimento, Martín Roffé, Vilma Regina Martins. Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B72.

Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex.

Phosphorylation negatively regulates exosome mediated secretion of cryAB in glioma cells

Exosomes mediate secretion of crystallin alphaB (cryAB), a well characterized molecular chaperone with anti-apoptotic activity. However, the mechanisms governing its packaging and secretion remained unexplored. In glioma cells, notwithstanding extensive phosphorylation of cryAB at Ser59 followed by Ser45 (Ser19 is largely unphosphorylated), we discovered that the majority of secreted exosomal cryAB is nonphosphorylated. Transient ectopic expression of a yellow fluorescent protein (YFP) tagged triple phosphomimic (3-SD) cryAB construct in cryAB absent glioma cells led to the formation of large cytosolic inclusions. Our findings demonstrate that mimicking phosphorylation significantly reduces cryAB secretion via exosomes. Moreover, decreased colocalization of 3-SD YFP-cryAB with multivesicular endosome (MVE) and exosome marker, CD63 or Rab27, a small GTPase regulating exocytosis of MVEs, suggests that phosphorylation deters packaging of cryAB in vesicles bound for secretion as exosomes. Additionally, we found that preventing O-GlcNAcylation on cryAB also curtailed its colocalization with CD63 and Rab27 resulting in reduced exosomal secretion. Thus, our study points to O-GlcNAcylation and lack of phosphorylation as being the selective processes involved in the packaging and secretion of cryAB via exosomes.

Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells.

Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation.

Cardiac CaM Kinase II genes δ and γ contribute to adverse remodeling but redundantly inhibit calcineurin-induced myocardial hypertrophy.

Ca(2+)-dependent signaling through CaM Kinase II (CaMKII) and calcineurin was suggested to contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus calcineurin for adverse cardiac remodeling remained unclear. We generated double-knockout mice (DKO) lacking the 2 cardiac CaMKII genes δ and γ specifically in cardiomyocytes. We show that both CaMKII isoforms contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2, and histone deacetylase 4, but also calcineurin. Under baseline conditions, DKO mice are viable and display neither abnormal Ca(2+) handling nor functional and structural changes. On pathological pressure overload and β-adrenergic stimulation, DKO mice are protected against cardiac dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411. Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. On exercise performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling. We established a mouse model in which CaMKII's activity is specifically and completely abolished. By the use of this model we show that CaMKII induces maladaptive cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of heart failure.

A novel role of proteasomal β1 subunit in tumorigenesis

p27Kip1 is a key cell-cycle regulator whose level is primarily regulated by the ubiquitin–proteasome degradation pathway. Its β1 subunit is one of seven β subunits that form the β-ring of the 20S proteasome, which is responsible for degradation of ubiquitinated proteins. We report here that the β1 subunit is up-regulated in oesophageal cancer tissues and some ovarian cancer cell lines. It promotes cell growth and migration, as well as colony formation. β1 binds and degrades p27Kip1directly. Interestingly, the lack of phosphorylation at Ser158 of the β1 subunit promotes degradation of p27Kip1. We therefore propose that the β1 subunit plays a novel role in tumorigenesis by degrading p27Kip1.

Abstract 575: Cdk1 prevents DNA rereplication in G2/M by phosphorylating and facilitating the removal of Cdc7 from chromatin at the end of S phase.

Abstract Cancer is inextricably linked with aneuploidy. Aneuploidy can arise for a number of reasons, including the deregulation of DNA and centrosome replication. Mechanisms have evolved that are designed to ensure that DNA is replicated only once per cell cycle. The initiation of DNA replication is a key target of these mechanisms, which includes regulating Cdc6, Cdt1 and Cyclin dependent kinases (Cdks). Although the regulatory mechanisms of Cdc6 and Cdt1 are known, the role of Cdk1 in particular is less clear. One potential target for Cdk1 regulation is the Dbf4-Cdc7 kinase (DDK), which acts as a licensing factor for DNA replication. DDK performs an essential and critical step in initiating DNA replication. As such, it is a target for novel cancer therapeutic drugs currently in clinical trials. Even so, the regulatory mechanisms dictating DDK function in human cells remains poorly understood. A site-directed mutagenesis approach was undertaken to determine the in vivo function of Cdk-dependent phosphorylation sites on the primary Cdc7 sequence. Using over-expressed Cdc7 in human cells (HEK293T), the functional significance of Cdc7 phosphorylation was determined; both the lack of phosphorylation and constitutive phosphorylation of Cdc7 was analyzed. We found that Cdc7, the catalytic subunit of the DDK, is a target of Cdk1. Cdc7 is phosphorylated in G2/M phase by Cdk1. Phosphorylation of Cdc7 removes the catalytic subunit of DDK from origins of replication and chromatin with the assistance of the nuclear export factor CRM1, which relocates Cdc7 to the microtubule cytoskeleton. Finally, the Cdk1-dependent removal of Cdc7 from chromatin prevents the cell from initiating DNA replication, indicating that Cdc7 is a target of Cdk1-dependent inhibition of DNA re-replication. We conclude that (1) Cdk1 phosphorylates Cdc7 in vivo. (2) Cdk1 dependent phosphorylation of Cdc7 regulates chromatin binding and DNA replication and (3) pCdc7 is associated with the nuclear export factor CRM1 in G2/M phase, suggesting a mechanism that couples the down-regulation of Cdc7 function at the end of S phase through nuclear localization and chromatin association. Taken together, our data provides key insight into the functional understanding of DDK. Citation Format: James Knockleby, Byung Ju Kim, Hoyun Lee. Cdk1 prevents DNA rereplication in G2/M by phosphorylating and facilitating the removal of Cdc7 from chromatin at the end of S phase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2013-575

Ser129D mutant alpha-synuclein induces earlier motor dysfunction while S129A results in distinctive pathology in a rat model of Parkinson's disease

Alpha-synuclein phosphorylated at serine 129 (S129) is highly elevated in Parkinson's disease patients where it mainly accumulates in the Lewy bodies. Several groups have studied the role of phosphorylation at the S129 in α-synuclein in a rat model for Parkinson's disease using recombinant adeno-associated viral (rAAV) vectors. The results obtained are inconsistent and accordingly the role of S129 phosphorylation in α-synuclein toxicity remains unclear. This prompted us to re-examine the neuropathological and behavioral effects of the S129 modified α-synuclein species in vivo. For this purpose, we used two mutated forms of human α-synuclein in which the S129 was replaced either with an alanine (S129A), to block phosphorylation, or with an aspartate (S129D), to mimic phosphorylation, and compared them with the wild type α-synuclein. This approach was similar in design to previous studies, however our investigation of dopaminergic degeneration also included performing a detailed study of the α-synuclein induced pathology in the striatum and the analysis of motor deficits. Our results showed that overexpressing S129D or wild type α-synuclein resulted in an accelerated dopaminergic fiber loss as compared with S129A α-synuclein. Furthermore, the motor deficit seen in the group treated with the mutant S129D α-synuclein appeared earlier than the other two forms of α-synuclein. Conversely, S129A α-synuclein showed significantly larger pathological α-synuclein-positive inclusions, and slower dopaminergic fiber loss, when compared to the other two forms of α-synuclein, suggesting a neuroprotective effect of the mutation. When examined at long-term, all three α-synuclein forms resulted in pathological accumulations of α-synuclein in striatal fibers and dopaminergic cell death in the substantia nigra. Our data show that changes in the S129 residue of α-synuclein influence the rate of pathology and neurodegeneration, with an overall deleterious effect of exchanging S129 to a residue mimicking its phosphorylated state.

Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target

One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.

N-Formyl Peptide Receptor 3 (FPR3) Departs from the Homologous FPR2/ALX Receptor with Regard to the Major Processes Governing Chemoattractant Receptor Regulation, Expression at the Cell Surface, and Phosphorylation

Among human N-formyl peptide chemoattractant receptors, FPR2/ALX and FPR3 share the highest degree of amino acid identity (83%), and trigger similar cell responses upon ligand binding. Although FPR2/ALX is a promiscuous receptor, FPR3 has only one specific high affinity ligand, F2L, and a more restricted tissue/cell distribution. In this study, we showed that FPR2/ALX behaved as the prototypical receptor FPR1. The agonist-dependent phosphorylation used a hierarchical mechanism with a prominent role of Ser(329), Thr(332), and Thr(335). Phosphorylation of FPR2/ALX was essential for its desensitization but the lack of phosphorylation did not result in enhanced or sustained responses. In contrast, resting FPR3 displayed a marked level of phosphorylation, which was only slightly increased upon agonist stimulation. Another noticeable difference between the two receptors was their subcellular distribution in unstimulated cells. Although FPR2/ALX was evenly distributed at the plasma membrane FPR3 was localized in small intracellular vesicles. By swapping domains between FPR2/ALX and FPR3, we uncovered the determinants involved in the basal phosphorylation of FPR3. Experiments aimed at monitoring receptor-bound antibody uptake showed that the intracellular distribution of FPR3 resulted from a constitutive internalization that was independent of C terminus phosphorylation. Unexpectedly, exchanging residues 1 to 53, which encompass the N-terminal extracellular region and the first transmembrane domain, between FPR2/ALX and FPR3 switched localization of the receptors from the plasma membrane to intracellular vesicles and vice versa. A clathrin-independent, possibly caveolae-dependent, mechanism was involved in FPR3 constitutive internalization. The peculiar behavior of FPR3 most probably serves distinct physiological functions that remain largely unknown.

Dynamic phosphorylation of AQP2 at S269 modulates AQP2 endocytosis

Phosphorylation of aquaporin-2 (AQP2) at S256 and S269 regulates plasma membrane accumulation of the protein. We aimed to distinguish the role of each site in regulation of AQP2. Forskolin treatment of wt-AQP2 MDCK cells increased pS269-AQP2, whereas levels of pS256-AQP2 did not change. Lack of phosphorylation at S269 (269A-AQP2) or mimicking phosphorylation at S269 (269D-AQP2) did not alter pS256-AQP2 levels. In wt-AQP2 cells, pS269-AQP2 rapidly disappeared in response to forskolin washout, whereas pS256-AQP2 levels remained high. In contrast to pS256-AQP2, following forskolin washout pS269-AQP2 was not observed in the internalized pool of AQP2. Combined with our previous observations that 269D-AQP2 has a decreased rate of internalization and is located in the membrane, these results emphasize a role of pS269 in membrane retention of AQP2. The double mutants 256D/269A-AQP2 and 256D/269D-AQP2 were located in the plasma membrane, whereas 256A/269A-AQP2 and 256A/269D-AQP2 were predominantly intracellular, suggesting that pS256 is dominant for determining AQP2 localization. The 256D/269A-AQP2 and 256D/269D-AQP2 double mutants had decreased rates of internalization compared to WT-AQP2, with 256D/269D-AQP2 being internalized slowest. In vitro kinase screening assays revealed that, in addition to PKA, AQP2 can be phosphorylated by other members of the AGC group.

Regulation of Serotonin 1B Receptor by Glycogen Synthase Kinase-3

In response to 5-hydroxytryptamine (5-HT), the type 1 serotonin receptors (5-HT1Rs) preferentially couple to the inhibitory G protein and elicit many physiological and behavioral processes. However, their regulation by intracellular protein kinases has not been fully investigated. In this study, we identified that glycogen synthase kinase-3 (GSK3) differentially regulates 5-HT1Rs. In receptor-expressing cells and brain slices, activation of both 5-HT1AR and 5-HT1BR reduced forskolin-stimulated cAMP production, but only the effect of 5-HT1BR was abolished by selective GSK3 inhibitors, deletion of GSK3beta by RNAi, or overexpression of impaired GSK3beta mutants (R96A and K85,86A). A consensus GSK3 phosphorylation sequence was identified between the serine-154 and threonine-158 in the second intracellular loop of 5-HT1BR. Mutation of either serine-154 or threonine-158 to alanine significantly reduced response of 5-HT1BR to 5-HT. Active GSK3beta interacted with resting 5-HT1BR to form a protein complex. The interaction was enhanced by receptor activation, abolished by GSK3 inhibitors, and dependent on the phosphorylation state of serine-154. In addition, regulation of 5-HT1BR by GSK3 changed the dynamics of agonist-induced cell surface receptor internalization, in which lack of phosphorylation at Ser154 resulted in sustained reduction of 5-HT1BR at the cell surface. Although the physiological consequences of selective regulation of 5-HT1BR by GSK3 remain to be identified, findings in this study reveal a new function of GSK3 as a protein kinase that is able to selectively regulate G protein-coupled receptors.

Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

BackgroundCell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process.Methodology/Principal FindingsHere, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology.Conclusions/SignificanceWe conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.

In Vivo Phosphorylation Site Mapping in Mouse Cardiac Troponin I by High Resolution Top-Down Electron Capture Dissociation Mass Spectrometry: Ser22/23 Are the Only Sites Basally Phosphorylated

Cardiac troponin I (cTnI) is the inhibitory subunit of cardiac troponin, a key myofilament regulatory protein complex located on the thin filaments of the contractile apparatus. cTnI is uniquely specific for the heart and is widely used in clinics as a serum biomarker for cardiac injury. Phosphorylation of cTnI plays a critical role in modulating cardiac function. cTnI is known to be regulated by protein kinase A and protein kinase C at five sites, Ser22/Ser23, Ser42/44, and Thr143, primarily based on results from in vitro phosphorylation assays by the specific kinase(s). However, a comprehensive characterization of phosphorylation of mouse cTnI occurring in vivo has been lacking. Herein, we have employed top-down mass spectrometry (MS) methodology with electron capture dissociation for precise mapping of in vivo phosphorylation sites of cTnI affinity purified from wild-type and transgenic mouse hearts. As demonstrated, top-down MS (analysis of intact proteins) is an extremely valuable technology for global characterization of labile phosphorylation occurring in vivo without a priori knowledge. Our top-down MS data unambiguously identified Ser22/23 as the only two sites basally phosphorylated in wild-type mouse cTnI with full sequence coverage, which was confirmed by the lack of phosphorylation in cTnI-Ala(2) transgenic mice where Ser22/23 in cTnI have been rendered nonphosphorylatable by mutation to alanine.