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Thrombin-induced phosphorylation of MARCKS does not alter its interactions with calmodulin or actin
B S Neltner1, Y Zhao, D B Sacks
1Department of Internal Medicine (Pulmonary and Critical Care Medicine), University of Cincinnati Medical Center, Cincinnati, OH 45267-0564, USA.
Abstract:
Myristoylated alanine-rich C kinase substrate (MARCKS) is a calmodulin (CaM)- and actin-binding protein and prominent protein kinase C (PKC) substrate. In vitro phosphorylation of MARCKS by PKC has been shown to induce the release of both CaM and actin, leading to the suggestion that MARCKS may regulate CaM availability during agonist-induced signalling. In support of this hypothesis we previously demonstrated that thrombin-induced MARCKS phosphorylation in endothelial cells (EC) parallels activation of myosin light chain kinase, a CaM-dependent enzyme. To test this theory further, we transfected CHO cells, which normally do not express significant levels of MARCKS, with a MARCKS cDNA. The thrombin-stimulated phosphorylation of myosin light chains and the sensitivity to CaM antagonists in the MARCKS overexpressing cells was the same as that in control CHO cells. MARCKS associated with the actin cytoskeleton in EC was markedly increased upon treatment with the PKC activator, PMA, but only modestly enhanced by thrombin treatment. Similarly, colocalisation of MARCKS with actin was enhanced when the EC were challenged with PMA but not thrombin. These data may be partially explained by PKC-independent phosphorylation of MARCKS in response to thrombin stimulation.
Insights
Myristoylated alanine-rich C kinase substrate (MARCKS) protein may not regulate calmodulin availability during cell signaling. Overexpressing MARCKS in cells did not alter responses to thrombin, suggesting other mechanisms are involved.
Area of Science:
- Cell biology
- Molecular signaling
- Biochemistry
Background:
- Myristoylated alanine-rich C kinase substrate (MARCKS) is a protein kinase C (PKC) substrate that binds calmodulin (CaM) and actin.
- Phosphorylation of MARCKS by PKC can release CaM and actin, suggesting MARCKS regulates CaM availability during signaling.
- Previous studies showed thrombin-induced MARCKS phosphorylation in endothelial cells (EC) correlates with CaM-dependent enzyme activation.
Purpose of the Study:
- To investigate the role of MARCKS in regulating CaM availability and cellular responses to agonist stimulation.
- To determine if MARCKS overexpression affects thrombin-induced signaling pathways in cells lacking endogenous MARCKS.
Main Methods:
- Transfection of Chinese hamster ovary (CHO) cells with MARCKS cDNA.
- Stimulation of cells with thrombin and phorbol 12-myristate 13-acetate (PMA).
- Analysis of myosin light chain phosphorylation, CaM antagonist sensitivity, and MARCKS-actin colocalization.
Main Results:
- MARCKS overexpression in CHO cells did not alter thrombin-stimulated myosin light chain phosphorylation or CaM antagonist sensitivity compared to controls.
- MARCKS association with the actin cytoskeleton increased upon PMA treatment in EC, but only modestly with thrombin.
- MARCKS colocalization with actin was enhanced by PMA but not by thrombin in EC, potentially due to PKC-independent phosphorylation.
Conclusions:
- MARCKS may not be the primary regulator of CaM availability during thrombin-induced signaling in endothelial cells.
- The differential effects of PMA and thrombin on MARCKS-actin interaction suggest complex regulation.
- PKC-independent phosphorylation of MARCKS might occur in response to thrombin stimulation.
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