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Proteomic Phosphosite Analysis Identified Crucial NPM-ALK-Mediated NIPA Serine and Threonine Residues.

ABSTRACT: Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma that shows in 60% of cases a translocation t(2;5)(p23;q35), which leads to the expression of the oncogenic kinase NPM-ALK. The nuclear interaction partner of ALK (NIPA) defines an E3-SCF ligase that contributes to the timing of mitotic entry. It has been shown that co-expression of NIPA and NPM-ALK results in constitutive NIPA phosphorylation. By mass spectrometry-based proteomics we identified nine serine/threonine residues to be significantly upregulated in NIPA upon NPM-ALK expression. Generation of phospho-deficient mutants of the respective phospho-residues specified five serine/threonine residues (Ser-338, Ser-344, Ser-370, Ser-381 and Thr-387) as key phosphorylation sites involved in NPM-ALK-directed phosphorylation of NIPA. Analysis of the biological impact of NIPA phosphorylation by NPM-ALK demonstrated that the ALK-induced phosphorylation does not change the SCFNIPA-complex formation but may influence the localization of NIPA and NPM-ALK. Biochemical analyses with phospho-deficient mutants elucidated the importance of NIPA phosphorylation by NPM-ALK for the interaction of the two proteins and proliferation potential of respective cells: Silencing of the five crucial NIPA serine/threonine residues led to a highly enhanced NIPA-NPM-ALK binding capacity as well as a slightly reduced proliferation in Ba/F3 cells.

SUBMITTER: Gengenbacher A

PROVIDER: S-EPMC6721280 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Proteomic Phosphosite Analysis Identified Crucial NPM-ALK-Mediated NIPA Serine and Threonine Residues.

Gengenbacher Anina A Müller-Rudorf Alina A Poggio Teresa T Gräßel Linda L Dumit Veronica I VI Kreutmair Stefanie S Lippert Lena J LJ Duyster Justus J Illert Anna L AL

International journal of molecular sciences 20190820 16

Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma that shows in 60% of cases a translocation t(2;5)(p23;q35), which leads to the expression of the oncogenic kinase NPM-ALK. The nuclear interaction partner of ALK (NIPA) defines an E3-SCF ligase that contributes to the timing of mitotic entry. It has been shown that co-expression of NIPA and NPM-ALK results in constitutive NIPA phosphorylation. By mass spectrometry-based proteomics we identified nine serine/threonine resi ...[more]

PMID: 31434245

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Project description:Mumps virus (MuV), a paramyxovirus containing a negative-sense nonsegmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel antiviral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced and mutating residue T at 101 to A (T101A) significantly enhanced activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis.Mumps virus (MuV) is a reemerging paramyxovirus that caused large outbreaks in the United States, where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. This work leads to a better understanding of viral RNA synthesis as well as to potential novel strategies to control mumps.

Dataset Information

Proteomic Phosphosite Analysis Identified Crucial NPM-ALK-Mediated NIPA Serine and Threonine Residues.

Publications

Proteomic Phosphosite Analysis Identified Crucial NPM-ALK-Mediated NIPA Serine and Threonine Residues.

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