Phosphorylation of Myosin-I from Rat Liver by Protein Kinase C Reduces Calmodulin Binding

doi:10.1006/bbrc.1995.2596

Biochemical and Biophysical Research Communications

Volume 216, Issue 1, 2 November 1995, Pages 90-102

https://doi.org/10.1006/bbrc.1995.2596 Get rights and content

Abstract

Three isoforms of the cytoskeletal-associated, mechanochemical enzymes known as myosin-I have been purified from rat liver; each coisolates with calmodulin. Incubation of the purified myosin-I′s with protein kinase Cγ and ³²P-ATP results in phosphorylation of the myosin-I heavy chains. After phosphorylation, the myosin-I isoforms bind less radiolabeled calmodulin in binding assays than observed for control samples. Since the purified isoforms are phosphoproteins as determined by immunoblotting with monoclonal antibodies which recognize phosphoamino acids, these results indicate that phosphorylation might play a role in regulation of myosin-I.

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Cited by (14)

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Calmodulin is an essential protein in the model organism Dictyostelium discoideum. As in other organisms, this small, calcium-regulated protein mediates a diversity of cellular events including chemotaxis, spore germination, and fertilization. Calmodulin works in a calcium-dependent or -independent manner by binding to and regulating the activity of target proteins called calmodulin-binding proteins. Profiling suggests that Dictyostelium has 60 or more calmodulin-binding proteins with specific subcellular localizations. In spite of the central importance of calmodulin, the study of these target proteins is still in its infancy. Here we critically review the history and state of the art of research into all of the identified and presumptive calmodulin-binding proteins of Dictyostelium detailing what is known about each one with suggestions for future research. Two individual calmodulin-binding proteins, the classic enzyme calcineurin A (CNA; protein phosphatase 2B) and the nuclear protein nucleomorphin (NumA), which is a regulator of nuclear number, have been particularly well studied. Research on the role of calmodulin in the function and regulation of the various myosins of Dictyostelium, especially during motility and chemotaxis, suggests that this is an area in which future active study would be particularly valuable. A general, hypothetical model for the role of calmodulin in myosin regulation is proposed.
Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine<inf>1A</inf> receptor at two distinct sites: Putative role in receptor phosphorylation by protein kinase C
2004, Journal of Biological Chemistry
Citation Excerpt :
Significant evidence suggests that phosphorylation of CaM-binding domains and the binding of CaM to a target protein can be mutually exclusive and in some cases antagonistic. Phosphorylation of target proteins by PKC has been shown to reduce binding of CaM to myosin 1 (50), the plasma membrane Ca2+ pump (51, 52), and calcium transport-like (CaT1) protein (53). Likewise, binding of CaM to neural tissue-enriched acidic protein (NAP-22) inhibits phosphorylation by PKC (54).
The serotonin 5-HT_1A receptor couples to heterotrimeric G proteins and intracellular second messengers, yet no studies have investigated the possible role of additional receptor-interacting proteins in 5-HT_1A receptor signaling. We have found that the ubiquitous Ca²⁺-sensor calmodulin (CaM) co-immunoprecipitates with the 5-HT_1A receptor in Chinese hamster ovary fibroblasts. The human 5-HT_1A receptor contains two putative CaM binding motifs, located in the N- and C-terminal juxtamembrane regions of the third intracellular loop of the receptor. Peptides encompassing both the N-terminal (i3N) and C-terminal (i3C) CaM-binding domains were tested for CaM binding. Using in vitro binding assays in combination with gel shift analysis, we demonstrated Ca²⁺-dependent formation of complexes between CaM and both peptides. We determined kinetic data using a combination of BIAcore surface plasmon resonance (SPR) and dansyl-CaM fluorescence. SPR analysis gave an apparent K_D of ∼110 nm for the i3N peptide and ∼700 nm for the i3C peptide. Both peptides also caused characteristic shifts in the fluorescence emission spectrum of dansyl-CaM, with apparent affinities of 87 ± 23 nm and 1.70 ± 0.16 μm. We used bioluminescence resonance energy transfer to show that CaM interacts with the 5-HT_1A receptor in living cells, representing the first in vivo evidence of a G protein-coupled receptor interacting with CaM. Finally, we showed that CaM binding and phosphorylation of the 5-HT_1A receptor i3 loop peptides by protein kinase C are antagonistic in vitro, suggesting a possible role for CaM in the regulation of 5-HT_1A receptor phosphorylation and desensitization. These data suggest that the 5-HT_1A receptor contains high and moderate affinity CaM binding regions that may play important roles in receptor signaling and function.
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Regular ArticlePhosphorylation of Myosin-I from Rat Liver by Protein Kinase C Reduces Calmodulin Binding

Abstract

Regular Article
Phosphorylation of Myosin-I from Rat Liver by Protein Kinase C Reduces Calmodulin Binding