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Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Protein phosphatase 2A activity associated with Golgi membranes during the G2/M phase may regulate phosphorylation of
Chad N Hancock1, Surabhi Dangi, Paul Shapiro
1Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA.
Abstract:
The extracellular signal-regulated kinase (ERK) 1 and 2 proteins are mitogen-activated protein kinase (MAPK) members that regulate cell proliferation and differentiation. ERK proteins are activated exclusively by MAPK kinase 1 and 2 phosphorylation of threonine and tyrosine residues located within the conserved TXY MAPK activation motif. Although dual phosphorylation of Thr and Tyr residues confers full activation of ERK, in vitro studies suggest that a single phosphorylation on either Thr or Tyr may yield partial ERK activity. Previously, we have demonstrated that phosphorylation of the tyrosine residue (Tyr(P) ERK) may be involved in regulating the Golgi complex structure during the G2 and M phases of the cell cycle (Cha, H., and Shapiro, P. (2001) J. Cell Biol. 153, 1355-1368). In the present study, we examined mechanisms for generating Tyr(P) ERK by determining cell cycle-dependent changes in localized phosphatase activity. Using fractionated nuclei-free cell lysates, we find increased serine/threonine phosphatase activity associated with Golgi-enriched membranes in cells synchronized in the late G2/early M phase as compared with G1 phase cells. The addition of phosphatase inhibitors in combination with immunodepletion assays identified this activity to be related to protein phosphatase 2A (PP2A). The increased activity was accounted for by elevated PP2A association with mitotic Golgi membranes as well as increased catalytic activity after normalization of PP2A protein levels in the phosphatase assays. These data indicate that localized changes in PP2A activity may be involved in regulating proteins involved in Golgi disassembly as cells enter mitosis.
Insights
Localized protein phosphatase 2A (PP2A) activity increases on Golgi membranes during mitosis. This suggests PP2A regulates Golgi disassembly by controlling tyrosine-phosphorylated extracellular signal-regulated kinase (ERK) levels.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Extracellular signal-regulated kinase (ERK) 1 and 2 are key regulators of cell proliferation and differentiation.
- ERK activation requires phosphorylation by MAPK kinase 1 and 2 within the TXY motif.
- Tyrosine-phosphorylated ERK (Tyr(P) ERK) has been linked to Golgi structure regulation during cell division.
Purpose of the Study:
- To investigate the mechanisms generating Tyr(P) ERK during the cell cycle.
- To determine cell cycle-dependent changes in localized phosphatase activity affecting ERK phosphorylation.
- To identify the specific phosphatase involved in regulating Tyr(P) ERK.
Main Methods:
- Cell synchronization to isolate specific cell cycle phases (G1 vs. late G2/early M).
- Fractionation of cell lysates to analyze Golgi-enriched membrane fractions.
- Phosphatase activity assays, inhibitor treatments, and immunodepletion assays to identify the responsible phosphatase.
- Quantification of PP2A association with Golgi membranes and its catalytic activity.
Main Results:
- Increased serine/threonine phosphatase activity was observed in Golgi-enriched fractions from late G2/early M phase cells compared to G1 phase cells.
- Protein phosphatase 2A (PP2A) was identified as the primary phosphatase responsible for this activity.
- Elevated PP2A association with mitotic Golgi membranes and increased PP2A catalytic activity were noted.
- These changes correlate with the generation of Tyr(P) ERK during mitosis.
Conclusions:
- Localized increases in PP2A activity on Golgi membranes occur during the G2/M transition.
- PP2A likely plays a role in regulating Golgi disassembly by modulating Tyr(P) ERK levels.
- This provides a mechanism for how cell cycle progression influences signaling pathways controlling organelle dynamics.
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