Cell signaling, Biochemistry

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

9.  Exocytosis of synaptic
vesicles involve 3 steps:
1. TEHTERING
2. DOCKING
3. FUSION

10. 2. Calcium signalling by ROC
 Receptor-operated channels (ROCs)
1. Nicotinic acetylcholine receptors
2. 5-HT5 [5-Hydroxytryptamine receptor or Serotonin Receptor]
3. AMPA receptors [α-Amino-3-hydroxy-5-methylisoxazole-4-
propionic acid receptor]
4. NMDA receptors [N-methyl-D-aspartate receptor]
5. P2X receptors

11. Mechanism of
Nicotinic
Acetylcholine
Receptors (Skeletal
Muscle
Contraction)

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.

14. ISOTYPE TYPES
Beta (β) PLC-β1, PLC-β2, PLC-β3, PLC-β4
Gamma (γ) PLC-γ1, PLC-γ2
Delta (δ) PLC-δ1, PLC-δ3, PLC-δ4
Epsilon (ε) PLC-ε1
Eta (ζ) PLC-ζ1, PLC-ζ2
Zeta (η) PLC-η1
Breakdown of:
Phosphatidylinositol – Activate Phospholipase C
Phosphatidylcholine – Activate Phospholipase D
 Isotypes of Phospholipase C:

15.  PLCβ-Isotype: Act
through G-Protein
coupled receptors
 PLCγ-Isotype: Act
through tyrosine
kinase receptors

16. Synthesis of DAG
& IP3 from
Phosphoinositol

17. Phospholipase C
Phosphoinositol
pyrophosphate
(PIP2)
Inositol 1,4,5-
triphosphate
(IP3)
IP3 gated calcium
release from ER
Activation of
calcium sensitive
intracellular
proteins
Diacylglycerol
(DAG)
Remains
embedded in the
plasma
membrane
Activation of
Protein Kinase C

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.

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.

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.

29. Functions of Protein Kinase C

30. Cell Responses in which GPCRs
Activate PLCβ
Target tissue Signal molecule Major response
Liver Vasopressin Glycogen
breakdown
Pancreas Acetylcholine Amylase secretion
Smooth muscle Acetylcholine Muscle contraction
Blood platelets Thrombin Platelet aggregation

31. Hormone-induced Cell Responses
Mediated by Cyclic-AMP
Target tissue Hormone Major Response
Thyroid gland Thyroid-stimulating hormone (TSH) Thyroid hormone synthesis and
secretion
Adrenal cortex Adrenocorticotrophic hormone (ACTH) Cortisol secretion
Ovary Luteinzing hormone (LH) Progesterone secretion
Muscle Adrenaline Glycogen breakdown
Bone Parathormone Bone resorption
Heart Adrenaline Increase in heart rate and force
of contraction
Liver Glucagon Glycogen breakdown
Kidney Vasopressin Water resorption
Fat Adrenaline, ACTH, Glucagon, TSH Triglyceride breakdown

32. 4. Calcium Signalling By Phospholipase D

33. Summary

34. Thank you !!!