Role of Ca2+ – calmodulin complex in cell signaling
- Calmodulin is a dumbbell shaped ‘calcium modulating’ protein that mediates most of the activities of Ca2+ ions.
- The word ‘calmodulin‘ means – cal(cium) + modul(ate) + in(g).
- Calmodulin is a ubiquitous eukaryotic regulator protein that is involved in many calcium-mediated processes (17kDa protein).
- Calmodulin is always found intracellularly.
- Calmodulin is expressed in many cell types and can have different subcellular locations (including the cytoplasm, within organelles, or associated with the plasma or organelle membranes).
Structure of calmodulin
- It is homologous to the muscle protein troponin C in structure and function.
- Calmodulin forms two globular domains connected by a flexible central linker.
- Each domain binds two calcium ions in E-F hand motifs (calmodulin can bind a total of four Ca2+ ions via EF hand motifs).
- The calcium binding sites are 12 amino acids long and contain many negatively-charged or polar amino acid residues such as aspartate, glutamate, and asparagine. Calmodulin is an acidic protein.
- The side chains on these amino acids form ionic bonds with the Ca2+
- Full occupancy of these calcium binding sites leads to a marked conformational change, which allows calmodulin to activate enzymes and ion channels.
When intracellular Ca2+ increases in response to some stimulus, calmodulin binds Ca2+. The binding of Ca2+ to calmodulin drives a conformational change in the protein which in turn activates the CaM kinase. The kinase then phosphorylates a number of target enzymes, regulating their activities.
Some other features of calmodulin
- Many of the proteins that Calmodulin binds are unable to bind calcium themselves, and use calmodulin as a calcium sensor and signal transducer.
- Calmodulin can also make use of the calcium stores in the endoplasmic reticulum, and the sarcoplasmic reticulum.
- Calmodulin can undergo post-translational modifications, such as phosphorylation, acetylation, methylation and proteolytic cleavage, each of which has potential to modulate its actions.
Ca2+/calmodulin dependent protein kinase (CaM kinase) – serine–threonine kinases
CaMKs are enzymes activated by increases in the concentration of intracellular calcium ions (Ca2+).
- On binding Ca2+/calmodulin, CaM kinase II activates itself by autophosphorylation.
- The degree of activation being dependent on the oscillation frequency of Ca2+
Function: Phosphorylation of serine and threonine residues on the receiving proteins
- Transfers phosphates from ATP to defined serine or threonine residues in other target proteins.
- These proteins then go on to activate downstream processes such as intracellular signalling, smooth muscle contractions, neurotransmitter and hormone synthesis and release, and cell cycle regulation
- Activated CaMK is involved in the phosphorylation of transcription factors and therefore, in the regulation of expression of responding genes.
There are two types of CaM kinase:
1) Specialized CaM kinases:
Example: myosin light chain kinase (Role in smooth muscle contraction)
- Myosin Light Chain (MLC) Kinase is activated by a calmodulin when it is bound by calcium ions.
- Activated MLC will phosphorylate the head of the myosin light chain which in turn cause smooth muscle contraction.
2) Multifunctional CaM kinases: (CaM kinase II)
Play a role in neurotransmitter secretion, transcription factor regulation, and glycogen metabolism.
Neurotransmitter secretion: Acetylcholine stimulation of G protein coupled receptors in secretory cells of the pancreas and parotid gland induces an IP3 mediated rise in Ca2+ that triggers the fusion of secretory vesicles with the plasma membrane and release of their contents into the extracellular space.
Glycogen metabolism: Activation of Phosphorylase kinase
Transcription factor regulation: phosphorylation of target transcription factors
Activation of specific transcription factors triggered by increased Ca2+ concentration.
Two mechanisms are there:
- Ca2+/calmodulin activate protein kinases that in turn phosphorylate transcription factors, thereby modifying their activity and regulating gene expression.
- Ca2+/calmodulin activate a phosphatase that removes phosphate groups from a transcription factor.
Example of this mechanism: Activation of T cells of the immune system
Ca2+ ions enhance the activity of an essential transcription factor, NFAT (nuclear factor of activated T cells).
- In unstimulated cells, phosphorylated NFAT is located in the cytosol.
- Following receptor stimulation and elevation of cytosolic Ca2+, the Ca2+/calmodulin complex binds to and activates calcineurin (a protein-serine phosphatase).
- Activated calcineurin then dephosphorylates key phosphate residues on cytosolic NFAT, exposing a nuclear localization sequence that allows NFAT to move into the nucleus and stimulate expression of genes essential for activation of T cells.
Other Functions of calcium – calmodulin complex:
Calmodulin mediates many crucial processes: inflammation, metabolism, apoptosis, smooth muscle contraction, intracellular movement, short-term and long-term memory, and the immune response.
The Ca2+/calmodulin complex also plays a key role in controlling the diameter of blood vessels and thus their ability to deliver oxygen to tissues.
- Endothelial cells contain a G protein–coupled receptor that binds acetylcholine and activates phospholipase C, leading to an increase in the level of cytosolic Ca2+.
- After Ca2+ binds to calmodulin, the resulting complex stimulates the activity of NO synthase (an enzyme that catalyzes formation of NO from O2 and the amino acid arginine).
- Because NO has a short half-life (2–30 seconds), it can diffuse only locally in tissues from its site of synthesis.
- In particular NO diffuses from the endothelial cell into neighboring smooth muscle cells, where it activates an intracellular NO receptor with guanylyl cyclase activity.
- The resulting rise in cGMP leads to activation of protein kinase G (PKG) which causes relaxation of the smooth muscle and thus vasodilation.
In addition to its effects on enzymes and ion transport, Ca2+/calmodulin regulates the activity of many structural elements in cells.
- The actin – myosin complex of smooth muscle (under β- adrenergic control)
- Various microfilament-mediated processes in non contractile cells (including cell motility, cell conformation changes, mitosis, granule release, and endocytosis).
- The Ca2+ – calmodulin complex can activate specific kinases. Two of them are shown here.
- These actions result in phosphorylation of substrates, and this leads to altered physiologic responses. This figure also shows that Ca2+ can enter cells through voltage- or ligand gated Ca2+
- The intracellular Ca2+ is also regulated through storage and release by the mitochondria and endoplasmic reticulum.
Role in glucose metabolism
- Activation of Phosphorylase kinase (in turn activate glycogen Phosphorylase) which leads to the release of glucose from glycogen catalysed by glycogen Phosphorylase.
Activation of calcium pumps
- One of the functions of the Ca2+/calmodulin complex is to activate calcium pumps.
- These pumps remove calcium from the cytoplasm by either pumping it out of the cell or storing it in the endoplasmic reticulum.
- By controlling the amount of calcium in the cell, the downstream responses are regulated.