Dataset Information

Dephosphorylation of major sperm protein (MSP) fiber protein 3 by protein phosphatase 2A during cell body retraction in the MSP-based amoeboid motility of Ascaris sperm.

ABSTRACT: The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward.

SUBMITTER: Yi K

PROVIDER: S-EPMC2710834 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Dephosphorylation of major sperm protein (MSP) fiber protein 3 by protein phosphatase 2A during cell body retraction in the MSP-based amoeboid motility of Ascaris sperm.

Yi Kexi K Wang Xu X Emmett Mark R MR Marshall Alan G AG Stewart Murray M Roberts Thomas M TM

Molecular biology of the cell 20090520 14

The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphata ...[more]

PMID: 19458186

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Project description:Previous studies have demonstrated that the JC virus (JCV) late regulatory protein agnoprotein is phosphorylated by the serine/threonine-specific protein kinase-C (PKC) and mutants of this protein at the PKC phosphorylation sites exhibit defects in the viral replication cycle. We have now investigated whether agnoprotein phosphorylation is regulated by PP2A, a serine/threonine-specific protein phosphatase and whether JCV small t antigen (Sm t-Ag) is involved in this regulation. Protein-protein interaction studies demonstrated that PP2A associates with agnoprotein and dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this interaction inhibited the dephosphorylation of agnoprotein by PP2A. The interaction domains of Sm t-Ag and agnoprotein with PP2A were mapped, as were the interaction domains of Sm t-Ag with agnoprotein. The middle portion of Sm t-Ag (aa 82-124) was found to be critical for the interaction with both agnoprotein and PP2A and the N-terminal region of agnoprotein for interaction with Sm t-Ag. To further understand the role of Sm t-Ag in JCV regulation, a stop codon was introduced at Ser90 immediately after splice donor site of the JCV early gene and the functional consequences of this mutation were investigated. The ability of this mutant virus to replicate was substantially reduced compared to WT. Next, the functional significance of PP2A in JCV replication was examined by siRNA targeting. Downregulation of PP2A caused a significant reduction in the level of JCV replication. Moreover, the impact of Sm t-Ag on agnoprotein phosphorylation was investigated by creating a double mutant of JCV, where Sm t-Ag stop codon mutant was combined with an agnoprotein triple phosphorylation mutant (Ser7, Ser11 and Thr21 to Ala). Results showed that double mutant behaves much like the triple phosphorylation mutant of agnoprotein during viral replication cycle, which suggests that agnoprotein might be an important target of Sm t-Ag with respect to the regulation of its phosphorylation. Collectively, these results suggest that there is an interplay between agnoprotein, Sm t-Ag and PP2A with respect to the regulation of JCV life cycle and this could be important for the progression of the JCV-induced disease, PML.

Dataset Information

Dephosphorylation of major sperm protein (MSP) fiber protein 3 by protein phosphatase 2A during cell body retraction in the MSP-based amoeboid motility of Ascaris sperm.

Publications

Dephosphorylation of major sperm protein (MSP) fiber protein 3 by protein phosphatase 2A during cell body retraction in the MSP-based amoeboid motility of Ascaris sperm.

Similar Datasets

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