Download Principles of cell signaling Lecture 2

Principles of cell signaling
Lecture 2
Johan Lennartsson
Molecular Cell Biology (1BG320), 2014
[email protected]
1
A cell senses signals from its surroundings via
receptor proteins
G protein-coupled
receptors
Enzyme-linked
receptors
TNF family
receptors
Ion channel
receptors
Nuclear
receptors
2
Phosphorylation is a post-translational modification
commonly used in signal transduction
Phosphate groups in proteins:
added by enzymes called kinases
carry two negative charges
can engage in hydrogen bond networks
stable against spontaneous hydrolysis
removed by enzymes called phosphatases
Phosphorylation is a chemically stable but reversible modification
and is therefore often used by the cell to regulate protein function
Phosphorylation
Phosphorylation occurs on hydroxyl groups (Ser, Thr &Tyr)
and is catalyzed by enzymes denoted kinases
Tyrosine (Tyr, Y)
Serine (Ser, S)
Threonine (Thr, T)
Kinases are group into:
tyrosine kinases
serine/threonine kinase
4
Phosphorylation
Mg2+
+
+
The phosphate group is donated from ATP (g-phosphate)
The negatively charged ATP is stabilized by interaction
with Lysines from the kinase protein and Mg2+ ions
Electrons on the hydroxyl group oxygen makes a
nucleophilic attack on the P atom, and ADP functions as
a leaving group
5
Dephosphorylation
Electrons on the Cys makes nucleophilic attack on P atom in
the phosphoprotein and the substrate protein functions as a
leaving group
Target protein
dephosphorylation
The Cys thiol group is regenerated by a nucleophilic attack by
a water molecule that is activated by glutamine residue in the
phosphatase
Phosphatase
regeneration
6
Phosphorylation regulates enzymatic activities
Inactive state
Active state
Electrostatic effects can cause structural changes in proteins
7
Phosphorylation regulates binding activities
The SH2 domain is a compactly folded
domain consisting if about 100 a.a.
In humans there are 120 different
SH2 domains within 115 proteins
Variations in the amino acids forming the second
socket in different SH2 domains allow them to bind
to phosphotyrosine adjacent to different sequences.
8
A cell senses signals from its surroundings via
receptor proteins
G protein-coupled
receptors
Enzyme-linked
receptors
TNF family
receptors
Ion channel
receptors
Receptor tyrosine kinases
Cytokine receptors
Nuclear
receptors
9
Receptor tyrosine kinases
Ligands for receptor tyrosine kinases are called growth factors
Growth factors act in endocrine, paracrine, juxtacrine and
autocrine fashion
Growth factors are important for proper development and
function of multicellular organisms since they participate in
cell – cell communication regarding cell proliferation, position
and identity (differentiation)
Many growth factors and components in the signal
transduction pathways they activate are proto-oncogenes
or tumor suppressor genes
10
Families of receptor tyrosine kinases
58 Receptor tyrosine kinases (RTKs) in 20 subfamilies
11
Receptor tyrosine kinases are activated by
ligand-induced dimerization
Transphosphorylation
(=autophosphorylation)
increased catalytic activity
docking sites for intracellular
signaling proteins
12
Receptor tyrosine kinase activation
Activation of RTKs: dimerization
14
EGF receptor dimerization
15
RTK dimerization places two kinase domains in
optimal position for autophosphorylation
16
Example: dimerization of c-Kit
c-Kit is a receptor tyrosine kinase
important for hematopoietic stem
cells, as well as melanocytes and
germ cells
Deregulated c-Kit activity has been
found in several types of cancer,
e.g. leukemia and GIST
The process of ligand-induced dimerization and kinase activation
has been well studied
17
Schematic structure of c-Kit
Extracellular domain: 5 Ig-like domains (D1-D5)
out of which D1-D3 binds ligand, SCF
Single transmembrane helix
Intracellular tyrosine kinase domain that
contains a kinase insert region important
for signal transduction
18
Early experiments indicating that c-Kit may
undergo dimerization upon SCF binding
Truncated receptors lacking the cytoplasmic domain
inhibit signaling
Antibodies against the cytoplasmic domain activate
the kinase domain
19
Western blotting (immunoblotting, WB, Ib)
20
Chemical cross-linking experiments suggested
ligand-induced c-Kit dimerization
Protein 2 Lys ..
Protein 1 Lys
Protein2-NH
..
HN-Protein 1
receptor dimer
receptor monomer
21
EMBO J. 1991 10(13):4121-8
The c-Kit ligand (SCF) is itself dimeric
In many cases growth factors are bivalent, i.e.
they have two identical binding surfaces
hydrophobic interactions
salt bridges at the periphery of binding surface
hydrogen bonds (H20-mediated)
Covalent disulfide bonds (Cys-S-S-Cys)
found in SCF
22
Proc. Natl. Acad. Sci. U. S. A. (2000) 97,:7732–7737
EMBO J (2000) 19, 3192–3203
c-Kit dimerization is driven by the bivalent
nature of its ligand (SCF)
EMBO J (2007) 26, 891–901
23
EMBO J (2000) 19:3192–3203
Proc. Natl. Acad. Sci. U. S. A. (2000);97:7732–7737
Interactions between D4 in the receptor monomers
are essential for the formation of a functional dimer
SCF
24
Proc. Natl. Acad. Sci. U. S. A. (2008) 105:7681-7686
The interactions between D4 (and D5) regions
bring the transmembrane close to each other
25
Cell (2007), 213-215
Dimerization places the two internal kinase domains
in optimal position for autophosphorylation
Note: one turn of the a-helix corresponds to 3.6 amino acids
Kinase
activity
+++
+++
+
++
+++
+/-
-
++
26
Mol. Biol. Cell. (2000) 11(10):3589-99
Alternative splicing affecting the rigid a-helical region may
produce receptor variants with different kinase activities
27
J. Biol. Chem., (2003) 278 (11): 9159-9166
Ligand-induced dimerization of c-Kit
The bivalent nature of SCF brings to c-Kit monomers into a dimeric
state (but inactive)
Interactions between the D4 (and D5) in the extracellular domains
brings the transmembrane helices (and intracellular kinase domains)
close to each other
Dimerization puts the kinase domains in an orientation relative each
other that is functional
28
Consequences of RTK activation
Autophosphorylation creates binding
sites for signal transduction molecules
(SH2 & PTB domains)
Bound signal transduction
molecules are activated
(localization & phosphorylation)
29
Src homology 2 (SH2) domain
The pocket of the SH2
domain is highly specific for
phospho-Tyr because of the
bulky size of the tyrosine.
The other side of the pocket is more variable and allows specific
recognition of the residues at the C-terminal of the phosphotyrosine
One side of the pocket is lined with conserved basic amino
acids and binds the phosphotyrosine
30
Src domains interact with phosphorylated
tyrosine residues (pTyr)
31
Attraction of SH2-domain containing proteins depends on
the amino acid sequences outside the kinase domain
32
Src homology 3 (SH3) domain
SH3 domains are important for constitutive protein-protein
interactions and they have an elongated binding cleft,
where hydrophobic pockets contact the proline-rich
peptide helix
Proline
Poly-Pro helix
Recognize proline rich
motifs with minimal
sequence PxxP, where
x is any amino acid
Specificity is maintained by
amino acids either amino (or
carboxyl) terminal to the prolines
Side view
top view
Signal transduction proteins often contain
multiple domains
About 80 interaction domains have been identified
and signaling proteins often contains several
What are the consequences for a signaling protein
to bind to the receptor?
34
Activation of signal transduction molecules
Conformational change:
catalytic domain changes into a shape optimal for
catalysis
Activation of signal transduction molecules
Translocation:
substrate and activator are become co-localized
at a particular region of the cell
Activation of signal transduction molecules
Phosphorylation:
Addition of phosphate group causes a conformational
change and/or promote complex formation
37
RTK inactivation
In order for the cell to respond in an appropriate manner
to a growth factor the signaling has to be terminated
This occurs both at the level of intracellular pathways
but also at level of the receptor
38
RTKs are often internalized through clathrincoated pits after their activation
Adaptors and structural proteins
forming the clathrin cage
Dynamin is a GTPase needed
for budding of the vesicle
Actin is a component of
cytoskeleton
39
Endocytosis through clathrin-coated pits
A coated pit and a coated
vesicle formed during receptormediated endocytosis
(TEMs)
40
Clathrin trimer interact and form a lattice
that surrounds the vesicle
41
There are many endocytic pathways
besides clathrin-coated pits
Independent of uptake mechanism, most vesicles fuse
early endosomes
endosome
with larger vesicles called early
Endocytic vesicles fuse with early endosome
Rab proteins (G-proteins) on the endocytosed vesicle
interact with proteins (Rab effector protein) on the
endosome
As the membranes come closer to each other there is
a energetic barrier against membrane fusion.
The energy barrier is caused by the hydrophilic
membrane surfaces are solvated by water and these
molecules have to be removed before fusion can take
place
SNARE proteins on the vesicle and target compartment
provide energy to overcome this energy barrier
43
Endocytic vesicles fuse with early endosome
SNAREs on the vesicle selectively binds to SNAREs on the
acceptor membrane
Starting from the N-terminal, the SNAREs interact and form
a zipper-like structure
This forces the membranes to make contact, leading to their fusion
The endosome is an important sorting station
Endosomal sorting
Recycle back to plasma membrane
multivesicular bodies (e.g. RTK)
45
Activated RTKs become ubiquitinated and
this is important for intracellular sorting
c-Cbl
RTK
c-Cbl
RTK
endocytosis,
endocytosis,
lysosomal
lysosomal
degradation
degradation
RTK
RTK
endocytosis,
endocytosis,
proteasomal
proteasomal
degradation
degradation
endocytosis,
signaling
signaling
?
?
Ubiquitinated receptors are sorted into multivesicular bodies
by ESCRT complexes on the endosomal surface
Cytoplasm
Ligand binding region
Endosomal lumen
47
Exp Cell Res. 2009 315(9):1619-26
Multivesicular bodies will form/fuse with lysosomes
Lysosomes are organells that have a low internal pH (<5)
and contain acid hydrolase enzymes (including proteases)
that degrade proteins and other cell debris
48
A cell senses signals from its surroundings via
receptor proteins
G protein-coupled
receptors
Enzyme-linked
receptors
TNF family
receptors
Ion channel
receptors
Receptor tyrosine kinases
Cytokine receptors
Nuclear
receptors
49
Cytokine receptors have no intrinsic kinase activity
but associate with Jak family of tyrosine kinases
Jak family of tyrosine kinases associated with cytokine receptors
(in non-stimulated cells in an inactive form)
Cytokine receptor dimerization/oligomerization leads to Jak
apposition leading transphosphorylation and activation
Active Jak:s tyrosine phosphorylate intracellular part of the
receptor on multiple sites
Four members in the Jak family: Jak1, Jak2, Jak3 and Tyk2
50
Cytokine receptors frequently contain protein
subunits shared with other receptor complexes
51
Since protein subunits are shared there is frequent
redundancy in cytokines signaling
52
A cell senses signals from its surroundings via
receptor proteins
G protein-coupled
receptors
Enzyme-linked
receptors
TNF family
receptors
Ion channel
receptors
Nuclear
receptors
53
G-protein coupled receptors (GPCR:s)
GPCR constitute a large family of cell surface receptors
(there are about 800 GPCRs in humans)
GPCR can function as monomer and dimers
Diversity through dimerization
54
G-protein coupled receptors (GPCR:s)
Based on available structural data on GPCR these conclusions
can be drawn regarding their achitecture:
Highly similiar transmembrane regions consisting of 7
helices (good basis for homology modeling of other
GPCRs for this region)
Ligandbinding
55
G-protein coupled receptors (GPCR:s)
The extracellular ligand-binding domain display larger
differences (consistent with the different types of ligands they
bind)
G-protein coupled receptors (GPCR:s)
In the cytoplasmic domain there are variations in the length and
secondary structure of the loops connecting the transmembrane
helices (this can explain G-protein selectivity)
57
G-protein coupled receptors (GPCR:s)
Ligand activated GPCRs utilize intracellular
heterotrimeric G-proteins to initiate signal transduction
58
G-protein coupled receptor activation
Ligand binding to GPCR result in conformational change in the
transmembrane helices that creats a G-protein binding surface on
the intracellular side
Ga
GTP
GDP
GTP
GTP
Binding of Ga subunit to the GPCR stimulate release of its bound
GDP
Since the concentration GTP is higher that GDP in the cytoplasm
this will most likely result in an exchange to GTP.
Ga-GTP dissociates from the receptor
59
G-protein coupled receptor activation
Active conformation
Inactive conformation
60
G-protein coupled receptor activation
61
Ga and Gbg subunits engage downstream effectors
62
The heterotrimeric G-protein cycle
The enzymatic activity of the Ga subunit is a GTPase activity
meaningthat it hydrolyses GTP into GDP, thus turning itself off.
The level of GDP vs GTP bound can be regulated by
interactions with other proteins
The heterotrimeric G-protein cycle
The GDP and GTP bound states of the Ga protein has
different conformations
Effector protein
The switch region(s) have
different conformations
depending on whether
GDP or GTP is bound to Ga
64
There are several types of Ga with different, sometimes
opposite, abilities to activate downstream targets
65
Termination of GPCR activity
Similar to that of RTK:s !
66
Downregulation of GPCR activity: b-arrestin
GRK
bg subunit
Signal tranduction
a subunit
GRK-mediated GPCR phosphorylation promotes the
binding of β-arrestin which result in:
targeting of many GPCRs for internalization in
clathrin-coated vesicles
uncoupling of GPCR from heterotrimeric G
proteins binding and activation
67
Downregulation of GPCR activity
GPCRs are removed from cell surface and degraded
The affinity of the GPCR for its ligand is in some cases
decreased when Ga subunit binds to GTP (= is active).
This will limit the number of Ga that can be activated.
Binding of Ga to adenylate cyclase will enhance the
intrinsic GTPase actvity of a-subunit leading to its
inactivation. This will limit the time adenylate cyclase is
active.
The PKA-induced substrate phosphorylation is transient
and in the absence of PKA activity will be
dephosphorylated. This will limit the signal produced
cAMP phosphodiesterases hydrolyze cAMP into AMP
and this will terminate the signal
68