Introduction to Calmodulin Binding Proteins

Latest Research Review

Danton H. O’Day, Mathavarajah, Sabateeshan, Michael A. Myre, Robert J. Huber, 2019. Calmodulin-mediated events during the life cycle of the amoebozoan Dictyostelium discoideum. Biological Reviews 00: 000-000; doi: 10.1111/brv.12573.

Open access:

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin-binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin-dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium.

Calmodulin-dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin-dependent transdifferentiation to re-establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium

The asexual life cycle restarts with the calmodulin-dependent germination of spores. Specific calmodulin-binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.

Previous Research

Calcium-mediated signal transduction represents the first evolved means of intracellular communication and is fundamental to the survival and functioning of all cells. The primary intracellular and essential target for calcium action is the small protein calmodulin (CaM). CaM cannot work alone but responds to calcium signals by binding to and regulating the function of other proteins. In spite of the importance of Ca2+ and CaM for growth and development in Dictyostelium, only a few CaMBPs have been characterized in any detail. Our lab has specifically been attempting to understand the role of calmodulin proteins in downstream events mediated by calcium signalling. Profiling using the Calmodulin (CaM) binding overlay technique (CaMBOT) has revealed that Dictyostelium discoideum possesses well over four-dozen calcium-dependent and -independent CaM-binding proteins (CaMBPs; O'Day, D.H., 2003. CaMBOT: profiling and characterizing calmodulin-binding proteins, Cell Signal. 15: 347-354). The CaMBPs that are present vary depending on the subcellular fraction and these patterns change during asexual fruiting body development as shown in the next figure (Gauthier, Mona L., and Danton H. O'Day, 2001. Detection of Calmodulin-Binding Proteins and Calmodulin-Dependent Phosphorylation Linked to Calmodulin-Dependent Chemotaxis to Folic and cAMP in Dictyostelium. Cell. Signal. 13: 575-584).

In spite of the central importance of calmodulin, the study of these target proteins is still in its infancy. Catalano and O’Day (2008) critically reviewed 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 was proposed.

Catalano, A. and D.H. O’Day, 2008. Calmodulin-binding proteins in the model organism Dictyostelium: A complete and critical review. Cellular Signalling 20: 277-291.

Calmodulin Binding Protein Profiles of Developmental Cell Fractions of Dictyostelium

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CaMBOT also is used to isolate cDNAs encoding CaMBPs from an expression library from mid-to-late multicellular development of Dictyostelium. The method for doing this is outlined in the following figure.

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Previously studied CaMBPs such as calcineurin A (CNA) and regulatory myosin light chain (RMLC) were identified in this way. Novel proteins were also identified including Nucleomorphin. Studies on two other novel CaMBPs are currently being completed.

A diversity of studies implicates CaM in a number of cellular and developmental events. In keeping with this, Dictyostelium is known to possess around 60 potential CaMBPs, many localize to specific subcellular locales that change depending on the cellular or developmental event in progress. In spite of this, only a few of these proteins have been characterized to any degree. Very few CaMBPs of Dictyostelium have been proven to bind CaM and only a subset of these have had their CaMBDs verified through the binding of deletion constructs to CaM. Reviewing the literature shows that CaM-binding generally has not been well addressed. While it is becoming common for researchers to indicate the presence of IQ motifs in proteins, the validity of these as true Ca2+-independent CaMBDs often remains unexplored. The identification of Ca2+-dependent and Ca+-independent CaMBDs is only a preliminary step in identifying a protein as a CaMBP. To date, none of CaMBDs within potential Dictyostelium CaMBPs have been proven to directly interact with CaM using crystallography or NMR. While Ca2+-dependent CaMBDs are comparatively less well defined than other protein–protein interaction motifs, scanning for potential CaMBDs serves as a primary step in CaMBD elucidation. The recent development of a discriminative method may be useful for scanning the genome of Dictyostelium to identify their potential CaMBPs and their binding domains. Coupling this with CaMBD analyses using the Calmodulin Target Database should significantly increase the chances of identifying true CaMBPs from genomic database screening. Approximately 80% of the CaMBDs identified using the Calmodulin Target Database turn out to be true CaM-binding motifs. This method has been used to design protein constructs in Dictyostelium for the determination of CaM-binding regions for nucleomorphin, DdPGK and DdTK. Since Ca2+ is a central regulator of many cellular and developmental processes in this classic model organism, the continued pursuit of the complexity, interaction, and functions of the CaMBP population is essential, if we truly hope to dissect how these Ca2+-dependent processes work.


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Last update:  January 3, 2020