Identification of in vivo phosphorylation sites of SET, a nuclear phosphoprotein encoded by the translocation breakpoint in acute undifferentiated leukemia

SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is identified as a 39-kDa phosphoprotein found predominantly in the cell nuclei [1994, J. Biol. Chem. 269,2258-2262]. SET is fused to a putative oncoprotein, CAN, in AUL and is thought to regulate the transformation potential of SET-CAN by its nuclear localization and phosphorylation. We investigated in detail the in vivo phosphorylation of SET. Phosphorylation of SET occurred in all human cell lines examined in vivo, primarily on serine residues. Endoproteinase Glu-C digestion of phosphorylated SET yielded two phosphopeptides. By radiosequencing, we identified the in vivo phosphorylation sites of SET as Ser⁹ and Ser²⁴. The surrounding sequences of Ser⁹ and Ser²⁴ contained an apparent consensus site sequence for protein kinase C.     

Analysis of nucleo-cytoplasmic shuttling of the proto-oncogene SET/I2PP2A

SET/I2PP2A is a nuclear protein that was initially identified as an oncogene in human undifferentiated acute myeloid leukemia, fused to the nuclear porin Nup-214. In addition, SET is a potent inhibitior of the phosphatase PP2A. Previously, we proposed a model in which the small GTPase Rac1 recruits SET from the nucleus to the plasma membrane to promote cell migration. This event represents an entirely novel concept in the field of cell migration. Now, fluorescent versions of the SET protein are generated to analyze its nucleo-cytoplasmic shuttling in live cells. Our studies showed that under steady-state conditions a fraction of the SET protein, which is primarily localized in the nucleus, translocates to the cytosol in an apparently random fashion. SET exiting the nucleus was also seen in spreading as well as dividing cells. We designed an image analysis method to quantify the frequency of nuclear exit of the SET proteins, based on 4D confocal imaging. This straightforward method was validated by analysis of SET wild-type and mutant proteins. This showed that the frequency of nuclear exit of a Ser-9 phosphomimetic mutant (S9E) is enhanced compared to wild-type SET or a S9A mutant. Thus, we have developed a novel method to analyze the nucleo-cytoplasmic shuttling of the proto-oncogene SET dynamics in live cells. This method will also be applicable to monitor dynamic localization of other nuclear and/or cytoplasmic signaling proteins.     

The protein SET regulates the inhibitory effect of p21(Cip1) on cyclin E-cyclin-dependent kinase 2 activity.

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1) has a dual role in the regulation of the cell cycle; it is an activator of cyclin D1-CDK4 complexes and an inhibitor of cyclins E/A-CDK2 activity. By affinity chromatography with p21(Cip1)-Sepharose 4B columns, we purified a 39-kDa protein, which was identified by microsequence analysis as the oncoprotein SET. Complexes containing SET and p21(Cip1) were detected in vivo by immunoprecipitation of Namalwa cell extracts using specific anti-p21(Cip1) antibodies. We found that SET bound directly to p21(Cip1) in vitro by the carboxyl-terminal region of p21(Cip1). SET had no direct effect on cyclin E/A-CDK2 activity, although it reversed the inhibition of cyclin E-CDK2, but not of cyclin A-CDK2, induced by p21(Cip1). This result is specific for p21(Cip1), since SET neither bound to p27(Kip1) nor reversed its inhibitory effect on cyclin E-CDK2 or cyclin A-CDK2. Thus, SET appears to be a modulator of p21(Cip1) inhibitory function. These results suggest that SET can regulate G(1)/S transition by modulating the activity of cyclin E-CDK2.     

Proteomic analysis of SET-binding proteins

The protein SET is involved in essential cell processes such as chromatin remodeling, apoptosis and cell cycle progression. It also plays a critical role in cell transformation and tumorogenesis. With the aim to study new SET functions we have developed a system to identify SET-binding proteins by combining affinity chromatography, MS, and functional studies. We prepared SET affinity chromatography columns by coupling the protein to activated Sepharose 4B. The proteins from mouse liver lysates that bind to the SET affinity columns were resolved with 2-DE and identified by MS using a MALDI-TOF. This experimental approach allowed the recognition of a number of SET-binding proteins which have been classified in functional clusters. The identification of four of these proteins (CK2, eIF2alpha, glycogen phosphorylase (GP), and TCP1-beta) was confirmed by Western blotting and their in vivo interactions with SET were demonstrated by immunoprecipitation. Functional experiments revealed that SET is a substrate of CK2 in vitro and that SET interacts with the active form of GP but not with its inactive form. These data confirm this proteomic approach as a useful tool for identifying new protein-protein interactions.     

Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

We previously reported that protein kinase D2 (PKD2) in T cells is promptly activated after T-cell receptor (TCR) stimulation and involved in the activation of interleukin-2 promoter and T cell death, and that one of its candidate substrate is SET protein, a natural inhibitor for protein phosphatase 2A (PP2A). In this study, we investigated the target amino acid residues of SET phosphorylated by PKD2 and the effects of phosphorylation of SET on PP2A phosphatase activity. In vitro kinase assay using various recombinant SET mutants having Ser/Thr to Ala substitutions revealed that Ser171 of SET is one of the sites phosphorylated by PKD2. Recombinant SET with phosphorylation-mimic Ser171 to Glu substitution reduced its inhibitory effects on PP2A phosphatase activity compared with Ser171 to Ala substituted or wild-type SET. In addition, knockdown of PKD2 in Jurkat cells by RNAi or treatment of human CD4⁺ T cell clone with the PKD2 inhibitor Gö6976 resulted in reduced PP2A activity after TCR-stimulation judged from phosphorylation status of Tyr307 of the catalytic subunit of PP2A. These results suggest that PKD2 is involved in the regulation of PP2A activity in activated T cells through phosphorylation of Ser171 of SET.     

Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle

The cDNA encoding a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147-residue polypeptide, which we termed CPI17, a 17-kDa PKC-potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC₅₀=0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non-muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.     

Apolipoprotein E and peptide mimetics modulate inflammation by binding the SET protein and activating protein phosphatase 2A

The molecular mechanism by which apolipoprotein E (apoE) suppresses inflammatory cytokine and NO production is unknown. Using an affinity purification approach, we found that peptide mimetics of apoE, derived from its receptor binding domain residues 130-150, bound to the SET protein, which is a potent physiological inhibitor of protein phosphatase 2A (PP2A). Both holo-apoE protein and apoE-mimetic peptides bound to the C-terminal region of SET, which is then associated with an increase in PP2A-mediated phosphatase activity. As physiological substrates for PP2A, the LPS-induced phosphorylation status of signaling MAPK and Akt kinase is reduced following treatment with apoE-mimetic peptides. On the basis of our previous report, in which apoE-mimetic peptides reduced I-κB kinase and NF-κB activation, we also demonstrate a mechanism for reduced production of inducible NO synthase protein and its NO product. These data provide evidence for a novel molecular mechanism by which apoE and apoE-mimetic peptides antagonize SET, thereby enhancing endogenous PP2A phosphatase activity, which reduces levels of phosphorylated kinases, signaling, and inflammatory response.     

Nuclear translocation of a leukocyte elastase Inhibitor/Elastase complex during staurosporine-induced apoptosis: role in the generation of nuclear L-DNase II activity

Using L1210 murine leukemia cells, we have previously shown that in response to treatment with drugs having different targets, apoptotic cell death occurs through at least two different signaling pathways. Here, we present evidence that nuclear extracts from staurosporine-treated cells elicit DNase II activity that is not detected in nuclear extracts from cisplatin-treated cells. This activity correlates with the accumulation of two nuclear proteins (70 and 30 kDa) which are detected by an anti-L-DNase II antibody. Partial purification of this DNase II activity suggests that the 30-kDa protein could be the nuclease responsible for staurosporine-induced DNA fragmentation. The 70-kDa protein is also recognized by an anti-elastase antibody, suggesting that it carries residues belonging to both L-DNase II and elastase. Since previous findings showed that L-DNase II was generated from the leukocyte inhibitor of elastase, we propose that the 70-kDa protein results from an SDS-stable association between these two proteins and is translocated from the cytoplasm to the nucleus during staurosporine-induced apoptosis.     

Antitumor antibiotic fostriecin covalently binds to cysteine-269 residue of protein phosphatase 2A catalytic subunit in mammalian cells

Fostriecin is a phosphate monoester with excellent antitumor activity against mouse leukemia, and it is a potent inhibitor of protein phosphatase (PP) 2A. This compound has been predicted to covalently bind to the Cys269 residue of the PP2A catalytic subunit (PP2Ac) at the α,β-unsaturated lactone via a conjugate addition reaction. However, this binding has not yet been experimentally proven. To confirm such binding, we synthesized biotin-labeled fostriecin (bio-Fos), which has an inhibitory activity against the proliferation of mouse leukemia cells. We showed that fostriecin directly binds to PP2Ac in HeLa S3 cells by pull-down assays using bio-Fos. Moreover, we directly demonstrated that fostriecin covalently binds to the Cys269 residue of PP2Ac by matrix assisted laser desorption/ionization time-of-flight mass spectrometry analysis. From these results, the inhibitory mechanism of fostriecin on PP2A activity is discussed.     

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 μM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.     

Nuclear inhibitor of protein phosphatase-1 (NIPP1) directs protein phosphatase-1 (PP1) to dephosphorylate the U2 small nuclear ribonucleoprotein particle (snRNP) component, spliceosome-associated protein 155 (Sap155)

Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Recent work has revealed essential roles for Ser/Thr phosphatases, such as protein phosphatase-1 (PP1), in splicing, but how these phosphatases are regulated is largely unknown. We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155 (spliceosome-associated protein 155), an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap155 dephosphorylation. C-terminally truncated NIPP1 (NIPP1-DeltaC) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -DeltaC resulted in slight and severe decreases in Sap155 hyperphosphorylation, respectively, and the latter was accompanied with inhibition of splicing. PP1 overexpression produced similar effects, whereas small interfering RNA-mediated NIPP1 knockdown enhanced Sap155 hyperphosphorylation upon okadaic acid treatment. NIPP1 did not inhibit but rather stimulated Sap155 dephosphorylation by PP1 in vitro through facilitating Sap155/PP1 interaction. Further analysis revealed that NIPP1 specifically recognizes hyperphosphorylated Sap155 thorough its Forkhead-associated domain and dissociates from Sap155 after dephosphorylation by associated PP1. Thus NIPP1 works as a molecular sensor for PP1 to recognize phosphorylated Sap155.     

The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis

The Mixed-Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.