Download Calcium/calmodulin-dependent protein kinase II

Intracellular
Vytášek 2009
2+
Ca
signalling
2+
Ca
• is a universal intracellular signalling molecule
(secondary messenger), which controls numerous
developmental and cellular pathways
• intracellular concentration of resting cell is 50100nM, activated cells have Ca2+ concentration
above 1M
• concentration changes can activate extremely
various signaling cascades not only in different
types of cells but also in different areas of the
same cell (e.g. Ca2+ elevation in nucleus of a
neurone activates gene transcription and at the
synapse causes neurotransmitter release)
Processes affected by changes in Ca2+
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Learning and memory
Contraction and relaxation
Membrane excitability
Cell motility
Cytoplasmic and mitochondrial metabolism
Synthesis of proteins and lipids
Cell cycle
Apoptosis
2+
Ca binding
cellular proteins
• These proteins are conformationaly modulated by
calcium binding
• They exist on two forms :
- apo-form without calcium at intracellular Ca2+
concentration 50nM (resting state)
- calcium-form with bound Ca2+ after elevating of
intracellular Ca2+ concentration up to 1000nM
(activated state after stimulus)
• The typical motif of polypeptide chain, by which
Ca2+ is bound and which modulates the
conformation of the appropriate protein, is
EF-hand motif
EF-hand motif of calcium
binding site
Proteins containing EF-hand
domain
• Calmodulin subfamily
calmodulin, troponin C, light chain of
myosin
• Calcineurin subfamily
calcineurin (phosphatase)
• Other subfamilies
calpain (proteolytic enzyme), calbindin
(storing cytosolic protein of Ca2+)
Calmodulin
• present in all cells of eukaryotes
• a small, acidic protein approximately 148 amino
acids long (17kDa)
• highly conservated amino acid sequence
• four EF-hand domains respectively four binding
sites for calcium
• binding of calcium modulates its conformation
and then Ca2+-calmodulin is able or disable of the
interaction with its target proteins and complex of
Ca2+-calmodulin-target protein exhibits different
functional properties than uncomplexed target
protein
The conformational changes of
calmodulin after binding of
calcium ion
Ca2+
Blue
circles
Proteins affected complexation
with Ca2+-calmodulin
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Protein kinases (CaMK)
Calcineurin (protein phosphatase 2B)
Adenylate cyclase
NO synthases
cAMP phoshodiesterase
Ion chanels
Receptors
Calcium/calmodulin-dependent protein kinases
CaMK enzyme family is activated by increase in
intracellular Ca2+ and tranfers phosphate from ATP
to serine or threonine residue on other proteins
Calcium/calmodulin-dependent protein
kinases (CaMK) with narrow specifity
• CaMLCK - myosin light chain kinase
phoshorylates myosin light chain and this
phoshorylation is sufficient for muscle contraction
The enzyme exists on two forms - SkCaMLCK in
skeletal muscle cells and SmCaMLCK is present
in smooth muscle cells as well as in various other
cells.
Both CaMLCKs are stimulated by binding Ca2+calmodulin but this binding can be inhibited in the
case of SmCaMLCK by its own phoshorylation
(caused by various other kinases)
CaMK with narrow specifity
• PhK - phosphorylase kinase
phosphorylates and activates glycogen
phosphorylase (increases glycogen degradation)
• eEF-2K (CaMKIII) - eukaryotic elongation
factor 2 kinase
phosphorylates Thr-56 in eEF-2 (this factor
promotes ribosomal translocation along mRNA
during translation) and thus inhibits its activity.
The activity of eEF-2K is increased by binding
Ca2+-calmudulin and down-regulated by its own
phosphorylation caused by various kinases
CaMK with broad specifity
• CaMKI - Calcium/calmodulin-dependent protein
kinase I
three isoforms are products of distinct genes. Several
substrates for this enzyme were described but its
physiological substrates and roles are still unknown
• CaMKII- Calcium/calmodulin-dependent protein
kinase II
Particularly important in the nervous system. It is
localized in pre- and postsynaptic compartment. Four
isoforms can phosphorylate various proteins in every
part of a cell. It posses also autoregulatory properties.
It is supposed that CaMKII plays inportant role on
learning and memory
CaMK with broad specifity
• CaMKIV - Calcium/calmodulin-dependent protein
kinase IV
It contains nuclear localization sequence and therefore
it is responsible for Ca2+-dependent phoshorylation of
various nuclear transcription factors (but also
phosphorylates cytosolic proteins).
Autophosphorylation inhibits its activity
• CaMKK - Calcium/calmodulin-dependent protein
kinase kinase
dramatically increases the activity of CaMKI and
CaMKIV after binding CaMKK to Ca2+-calmodulin and
subsequent phosphorylation of CaMKI and CaMKIV
Calcineurin (protein phosphatase-2B)
• Heterodimeric molecule with highly conserved
domain structure
• Iron-zinc active catalytic center
• Dual protein phosphatase - dephosphorylates both
phosphoserine/threonine and phosphotyrosine
• Binding sites for calcium (four EF-hand domains
in calcineurinB chain)
• Preferentially dephosphorylates peptides with
basic AA on N-terminal end and without acidic
AA on C-terminus
• Main substrate of calcineurin is NFAT (nuclear
factor of activated T cells)
red sphere - calcium
gray and blue spheres - iron and zinc
yellow ribon - CnA
blue ribon - CnB
Calcineurin (protein phosphatase-2B)
• The main function is the regulation of gene expression
• Small activation is observed after increasing Ca2+
• High activation after supplementation and
complexation with Ca2+-calmodulin , increase in a 50100-fold of the Vmax . Afinity constant of this complex
is more than 1010.
• Its nhibitor is cyclosporin A (immunosupresory drug)
• The gene activation pathway on T cells is increase
Ca2+ - increased activity of Cn - dephosphorylation of
NFAT in cytoplasma - transport of the complex
NFAT-Cn to the nucleus - induction of cytokine genes
(IL-2,3,4 , TNF-a, GM-CSF)
Adenylyl cyclase
• enzyme synthetizing cAMP (the first identified
secondary messenger )
• nine isoforms are bound in plasma membrane and
single isoform is found in cytoplasma
• the activity of five membrane isoforms are
influenced also by calcium
• two isoforms 5 and 6 are inhibited by elevating
intracellular Ca2+ concentration
• two isoform 1 and 8 are activated by binding
complex calcium-calmodulin
• isoform 3 is inhibited by complex calciumcalmodulin-calmodulin kinase
The isoform-specific regulation
of adenylyl cyclases by calcium
NO synthases (NOS)
• Generates nitric oxide from L-arginine
• Three types of NOS are known nNOS,
iNOS a eNOS
• nNOS and eNOS are constitutively
synthetized and they are known as calciumdependent (its enzymatic activity is
determined by calcium)
• All three types can bind to calmodulin, but
nNOS and eNOS are activated only by
calmodulin with bound Ca2+.
2+
Ca -calmodulin
binding to nNOS
Modulation and effects of intracellular Ca2+
Increasing of the cytoplasmatic
2+
Ca concentration
1. Transport of Ca2+ from extracellular space transport can be mediated by calcium channel
gated by agonist (e.g. hormone) or by voltagegated channel
2. Liberating of Ca2+ from bound calcium in
endoplasmatic/sarcoplasmatic reticulum - ER/SR
are relatively abundant in calcium-binding
proteins (class I and II) and Ca2+ is liberated on
the cytoplasma after stimulus by IP3. (it is created
from phosphatidylinositoldiphosphate by cleavage
under catalysis of enzyme phospholipase C)
Signaling pathways of Ca2+
Oscillation of intracellular Ca2+
concentration in the various
types of cells