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Phosphorylation at the nuclear localization signal of Ca2+/calmodulin-dependent protein kinase II blocks its nuclear
E K Heist1, M Srinivasan, H Schulman
1Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5125, USA.
Abstract:
Translocation of protein kinases with broad substrate specificities between different subcellular compartments by activation of signaling pathways is an established mechanism to direct the activity of these enzymes toward particular substrates. Recently, we identified two isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), which are targeted to the nucleus by an alternatively spliced nuclear localization signal (NLS). Here we report that cotransfection with constitutively active mutants of CaM kinase I or CaM kinase IV specifically blocks nuclear targeting of CaM kinase II as a result of phosphorylation of a Ser immediately adjacent to the NLS of CaM kinase II. Both CaM kinase I and CaM kinase IV are able to phosphorylate this Ser residue in vitro, and mutagenesis studies suggest that this phosphorylation is both necessary and sufficient to block nuclear targeting. Furthermore, we provide experimental evidence that introduction of a negatively charged residue at this phosphorylation site reduces binding of the kinase to an NLS receptor in vitro, thus providing a mechanism that may explain the blockade of nuclear targeting that we have observed in situ.
Insights
Nuclear targeting of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is blocked by phosphorylation. CaM kinase I and IV phosphorylate a specific Ser residue, preventing CaM kinase II nuclear import.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Protein kinases translocate between cellular compartments to regulate substrate activity.
- Two Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) isoforms are known to be nuclear-targeted via an alternatively spliced nuclear localization signal (NLS).
Purpose of the Study:
- To investigate the mechanism by which nuclear targeting of CaM kinase II is regulated.
- To identify signaling pathways that modulate CaM kinase II localization.
Main Methods:
- Cotransfection with constitutively active CaM kinase I or CaM kinase IV mutants.
- In vitro kinase assays to assess phosphorylation of CaM kinase II.
- Mutagenesis studies to analyze the role of a specific phosphorylation site.
- In vitro binding assays to measure kinase-receptor interactions.
Main Results:
- Constitutively active CaM kinase I or CaM kinase IV specifically blocked nuclear targeting of CaM kinase II.
- Phosphorylation of a Ser residue adjacent to the NLS of CaM kinase II was identified as the mechanism.
- This phosphorylation was both necessary and sufficient to block nuclear targeting.
- Introduction of a negative charge at the phosphorylation site reduced CaM kinase II binding to an NLS receptor.
Conclusions:
- Phosphorylation of CaM kinase II by CaM kinase I or IV regulates its nuclear import.
- This regulatory mechanism involves modulating the interaction between CaM kinase II and its NLS receptor.
- Provides insight into the dynamic control of kinase localization in cellular signaling.