Signal transduction processes connected to the changes in cytosolic calcium concentration (calcium signaling)
1. Cell signalling due to
change in cytosolic Ca2+
concentration.
Protein Kinase C
PRADEEP SINGH
M.Sc. MED. BIOCHEMISTRY
HIMSR, JAMIA HAMDARD
2. Content
Introduction
Calcium Signalling
1. Ca2+ signalling by Voltage Operated Channels
2. Ca2+ signalling by Receptor Operated Channels
3. Ca2+ signalling by Hormones (Activation of Phospholipase)
A. Phospholipase C (IP3 & DAG)
Activation of Protein Kinase C
B. Phospholipase D
3. Introduction
Ca2+ is an essential element which regulate large number of
physiological processes such as proliferation, neural signaling,
learning, contraction, secretion, and fertilization.
Concentration of calcium varies greatly in intracellular and
extracellular environment.
Extracellular concentration of calcium is 10-3 M/L while the
intracellular concentration 10-7 M/L [10000 times lower than ECF].
So, regulation of Ca2+ levels in the cell is very important.
4. In mitochondria, increase in the concentration of free Ca2+ in the mitochondrial
matrix accelerate pyruvate oxidation and ATP production.
In muscle, increases in the concentration of Ca2+ are used both to induce
contraction and to coordinately increase mitochondrial ATP synthesis to provide
the energy for contraction.
5. Calcium Signalling
Calcium signalling pathway fall into two main groups
depending on how they are activated:
1. External Stimuli
2. Internal Stimuli
6. Calcium signalling by external stimuli takes place by
following pathways:
1. Calcium signalling by VOC (Voltage operated calcium)channels
2. Calcium signalling by ROC (Receptor operated calcium) channels
3. Calcium signalling by PLC (Phospholipase C)
4. Calcium signalling by PLD (Phospholipase D)
7. 1. Ca2+ Signalling by VOC
Secretory vesicles wait near the plasma Membrane (neurolemma)
until signaled to release their contents.
Membrane depolarization in the presynaptic membrane activate a
specific isoform of VOC.
VOC in the presynaptic endings are associated with the synaptic
vesicles, thus producing a highly localized puff of Ca2+ to trigger
exocytosis.
8. Exocytosis of Synaptic vesicles
Exocytosis of synaptic vesicles involve 3 steps:
1. TEHTERING
2. DOCKING
3. FUSION
Exocytosis of vesicles require 3 major proteins:
1. SNARE Proteins [t-SNARE, v-SNARE &
Synaptotagmin]
2. Rab-GTPase
3. Rab Effector Proteins
12. Steps of skeletal muscle contraction
1. Release of ACh at the neuromuscular junction
2. Generation of Motor End Plate potential
3. Opening of Dihydropyridine Receptors (Influx of Extracellular Ca2+)
4. Release of Calcium from Sarcoplasmic Reticulum by ryanodine
receptors (via physical coupling to the dihydropyridine receptors)
5. Ca2+ binds to troponin; blocking action of tropomyosin released
6. Contraction via crossbridge formation; ATP hydrolysis
7. Removal of Ca2+ by active transport
8. Tropomyosin bloackage restored; contraction ends
13. 3. Calcium Signalling by Phospholipase C
PLCs are a family of enzymes that hydrolyze a phosphoester bond in
certain phospholipids (Phosphatidylinositol & Phosphatidylcholine).
Breakdown of phospholipids yields two second messengers DAG & IP3.
DAG & IP3 function in elevating both the cytosolic and mitochondrial-
matrix Ca2+ levels.
Elevated Cytosolic Ca2+ levels and activate a family of cytosolic kinases
known as protein kinases (PKC & PKD)
Protein kinases in turn affect many important cellular processes such as
growth and differentiation as well as altering the activity of many
proteins.
18. A) IP3 Induced formation of calcium
calmodulin complex
IP3 induced release of calcium from the ER leads to 10-20 fold increase in
cytosolic calcium concentration.
Various Ca2+ binding protein acts as calcium buffers and restrict the diffusion
of increased cytosolic calcium to ECF.
Calcium binding proteins includes troponin, calbindin, calmodulin and
calcineurin.
19. The calcium ion oscillations occur in the pituitary gland cells that secrete luteinizing
hormone (LH), which plays an important role in controlling ovulation and thus female
fertility.
LH secretion is induced by the binding of luteinizing hormone-releasing hormone
(LHRH) to its G protein–coupled receptors on the surfaces of pituitary cells.
The signal often remains localized to the site
where the Ca2+ enters the cytosol.
Spikes of calcium concentration controls the gene
expression i.e., one frequency of Ca2+ spikes
activates the transcription of one set of genes,
while a higher frequency activates the
transcription of a different set of genes.
20. Ca2+/Calmodulin
Complex
Calmodulin consist of a highly
conserved single polypeptide
chain having two globular ends
which resembles dumbbell
shape.
Each globular head has 2 Ca2+
binding sites.
Binding of Ca2+ with calmodulin
induces conformational change
in the calmodulin which leads to
formation of Ca2+/Calmodulin
complex.
21. Functions of Ca2+/Calmodulin
complex
Ca2+/Calmodulin
complex
Ca2+/Calmodulin
complex bind to various
target proteins in the
cell to alter their activity
Ca2+/Calmodulin complex
activates the plasma membrane
Ca2+ pump that uses ATP to
pump Ca2+ out of the cells.
1.Ca2+/Calmodulin
complex activates
Ca2+/Calmodulin-
dependent kinases such
as CaM-kinase II
22. Ca2+/Calmodulin-Dependent Kinases (CaM-Kinase II)
CaM-kinase II is one of the most studied Ca2+/Calmodulin-dependent
Kinase.
CaM-kinase II plays an important role in learning and memory.
CaM-kinase II protein has two major domains: an amino terminal kinase
domain and a carboxy-terminal hub domain, linked by regulatory
protein.
23.
24. The complete enzyme contains two stacked rings around the central hub, for a
total of 12 kinase proteins (one ring of 6 kinase proteins on both side of the hub
domain).
In inactive state, the regulatory subunit is buried in the active site of the kinase
thereby blocking its catalytic activity.
When a kinase domain has popped out from the central hub domain, the
regulatory subunit is now accessible to the Ca2+/Calmodulin complex.
25. Ca2+/Calmodulin complex (if present) will bind the regulatory
segment and prevent it from inhibiting the kinase thereby activates
the kinase activity.
If the adjacent kinase subunit also pops out from the hub, it will
also be activated by Ca2+/Calmodulin complex and the two kinases
will then phosphorylate each other on their regulatory segment.
It converts the enzyme to Ca2+ independent form which activates
other kinases, so that the kinase remain active even after
dissociation of Ca2+/Calmodulin complex.
26. B) DAG based activation of Protein Kinase C
After the formation by phospholipase C, the hydrophobic DAG remains
associated with the plasma membrane.
The principle function of DAG is to activate Protein Kinase C (PKC).
In the absence of hormone stimulation, protein kinase C is present as a
soluble cytosolic protein that is catalytically inactive.
Increase in cytosolic Ca2+ levels causes protein kinase C to translocate to
cytosolic leaflet of plasma membrane, where it can interact with membrane
associated DAG.
27.
28. The activation of PKC in different cells plays an important role in
many aspects of cellular growth and metabolism.
PKC phosphorylates transcription factors that are localized in the
cytosol, triggering their movement into the nucleus, where they
activate genes necessary for cell division.
In liver cells, PKC helps regulate glycogen metabolism by
phosphorylating and so inhibiting glycogen synthase.
Ca2+ increased the conductance of Complex IV (2.3-fold), Complexes I and III (2.2-fold), ATP production/transport (2.4-fold), and fuel transport/dehydrogenases (1.7-fold).
External Stimuli (function to transfer information from the cell surface to internal effector system)
Internal Stimuli [Respond to information generated within the cell]
Cyclic ADP-Ribose/NAADP
Example – exocytosis of vesicles from presynaptic membrane
‘t’ means target membrane
‘v’ means vesicle membrane
Three types of proteins are involved in synaptic docking:
SNARE Protein (t-SNARE & v-SNARE) “t” snare means snare protein which is present at target membrane & “v” snare means snare protein which is present at the vesicle membrane.
2. RAB-GTPase – Present on vesicle membrane. This protein helps in the recognition of the target membrane.
3. RAB Effector Protein
Rab
Arrival of the nerve impulse at the neuromuscular junction.
Calcium induced calcium release from the ER in skeletal muscle cells
G protein called Gaq activates the inositol phospholipid signalling pathway. The activated phospholipase ten cleaves the Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to generate two products
Pi-4 kinase introduces a phosphate group at the 4th position.
Protein Kinase C is activated by combination of DAG, Ca2+ & negatively charged membrane phospholipid phosphatidylserine. Protein Kinase C in turn phosphorylates target proteins that vary depending on the cell type.
Ca2+/Calmodulin has no enzymatic activity itself but it acts by binding to and activating other proteins.
Ca2+/Calmodulin complex itself has no enzyme activity.
Front
Back
Complete enzyme contains two stacked rings of 12 CaM-kinase II proteins. Each ring contain 6 CaM-kinase II proteins on both sides of the hub domain. In inactive state, the regulatory subunit is buried in the active site of the kinase thereby blocks the kinase activity. When kinase domain has popped out and linked to the central hub by regulatory subunit. If present
Ca2+/Calmodulin complex binds the regulatory subunit of CaM-Kinase II
Activation of PKC thus depends on an increase of both Ca2+ ions and DAG.
Phosphatidylcholine breaks into Phosphatidic Acid & Choline.
Lyso PC = Lysophospholipids