1. fibroblast chemotaxis: more about positive feedback loops
DESCRIPTION
1. Fibroblast Chemotaxis: more about positive feedback loops. 2. Autoregulatory Mechanisms of Eukaryotic Chemotaxis System Components: Receptors, G-proteins, GEFs, PI3K, Kinases, phosphatases. How evolution has selected for components with autoregulation and integral feedback control. - PowerPoint PPT PresentationTRANSCRIPT
1. Fibroblast Chemotaxis: more about positive feedback loops.
2. Autoregulatory Mechanisms of Eukaryotic Chemotaxis System Components: Receptors, G-proteins, GEFs, PI3K, Kinases, phosphatases. How evolution has selected for components with autoregulation and integral feedback control.
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Fibroblasts chemotax toward growth factors
0
3 hrs
8 hrs
12 hrs
21 hrs
PDGF-stimulated wound healing in mouse embryo fibroblasts
PI3K p110 Family Members
110
110
110
110
KinasePIKC2Rasbinding
p85binding
Class Ia
Class Ib
Tissue
All
All
BloodCells
Blood Cells
Regulation
Tyr Kinase
Tyr Kinase + Tyr Kinase
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0
3 hrs
8 hrs
12 hrs
21 hrs
WT PI3K-Ia Deletion
Deletion of Class Ia PI3K genes in mouse embryo fibroblasts impairs PDGF-dependent cell migration.Brachmann et al., 2005 Mol. Cell. Biol. 25, 2593.
Woundhealing 10ng/ml PDGF
0
10
20
30
40
50
60
70
80
PI3K Ia deletion
Mig
rate
d C
ells
3h 8h 15h 21h
P85-/-;p85-/-Wild type
Ly294003PI3K inhibitor
unstimulated
PI3K Iadeletion
PDGF PDGF + WM
Wild Type
Defect in PDGF-induced lamellipodia formationin MEFs defective in class Ia PI3K
Brachmann et al., 2005 Mol Cell Biol 25, 2593
SH3 Cdc42binding
SH2 SH2
Membrane
CatalyticRasBinding
GTPRas
GTPCDC42 P-TyrP-Tyr
Class Ia PI 3-Kinase
PIK
Tyr Kinase
C2
Class Ia PI3K has multiple domains for signal input, allowing it to act as an ‘AND GATE’ or possibly an ‘OR GATE’
p85 regulatoryp110 catalytic
GPCR
p110 can also be activated by subunits of G proteins, but only when bound to a phosphoTyr protein (AND GATE).
AKT
PH
PIP3 PIP3
Class Ia PI3K mediates growth factor-dependent cortical actin formation
TyrKinase p110
PI3Kp85SH2
Receptor
GrowthFactor
P-Tyr
GTPRas
RacGEF?
PTEN
GTPRac
Cell Migration
Cortical Actin
PIP2
Wild Type MEFs PI3K Ia deleted
5 min PDGF [ng/ml]: 0 1 3 10 0 1 3 10
Erk-P
Erk
Deletion of class Ia PI3K genes appears to impair (but not eliminate) Ras activation (as judged by impaired activation of
the downstream protein kinase, Erk)Brachmann et al., 2005 Mol Cell Biol. 25, 2593
Thus, as in Dictyostelium, there appears to be a positive feedback loop between PI3K and Ras in fibroblasts.
Erk-P
Erk
Control Double KO
PDGF: - + - +
Rac GTP
Rac
p85
GST-CRIB pulldown
Reduced PDGF-induced Rac activationin MEFs lacking class Ia PI3K
Brachmann et al., 2005 Mol Cell Biol. 25, 2593
Overexpression of a Rac GEF (Vav2) induces lamellipodia formationin MEFs lacking Class Ia PI3K
Brachmann et al., 2005 Mol Cell Biol. 25, 2593
Rhodamine-Phalloidin
(Actin)
Vav2
Wild Type PI3K Ia deleted
PI3K is involved in both local Ras and local Rac positive feedback loops
+
+
RacGTP
p85 p110 PIP3
GEFPH
?
RasGTP
?PDGF
Receptor
Conclusions1. Growth Factor Receptors stimulate Class Ia PI3K through
PhosphoTyr residues of receptors binding to SH2 domains, while GPCRs stimulate Class Ib PI3K through subunits binding to the catalytic subunit.
2. In both cases, PI-3,4,5-P3 is in a local positive feedback amplification loop involving Rac (and Ras?) that allows non-isotrophic localization of cortical actin, providing directionality to chemotaxis.
How is perfect adaptation achieved in eukaryotic chemotaxis?
Shutoff mechanisms must exist to adapt the system to a given level of stimulation, allowing a temporal increase in receptor stimulation to be sensed. The adaptation should be slow compared to the stimulation to insure significant directional migration prior to adaptation.
What is known about shutoff mechanisms of GPCRs and Receptor Tyr Kinases?
Receptor
GDP
Hormone
Receptor
GDP
GPCR ACTIVATION
Receptor
GDP
attractant
Receptor
Effector 2
PI 3-kinase etc.
Ligand-inducedConformationalChange <100 msec
GDP GTP
Receptor
GTP
GTP
Receptor
Effector 1
(Phospholipase C, etc.)
msec to sec
GPCR ACTIVATION
Receptor
GTP
Signal Termination, Downregulation and Reset to Basal State
Effector 1
Effector 2
Minutes
GDP
RGSSeconds
Receptor
GTP
Signal Termination, Downregulation and Reset to Basal State
Effector 1
Minutes
GDP
RGSSeconds
G-ReceptorKinase (GRK)
Receptor
GTP
Signal Termination and Reset to Basal State
G-ReceptorKinase (GRK)
P P P
Receptor
P P P
Inactive
Minutes
GDP
RGSSeconds
Arrestin
Receptor
DephosphorylationAnd rebinding ofG and . (minutes)
GDP
Basal State
Effector 1
Phosphatase
Only activated receptors are phosphorylated and downregulated. This effect is slow (minutes) compared to activation (seconds). During this perturbation from steady state, PI3K activation occurs, driving directional motility.
Integral Feedback ControlAnalogous to model in Yi, Huang, Simon&Doyle 2000 PNAS 97, 4649
If we assume that only activated receptors are phosphorylated (and thus inactivated) and that the phosphatase that dephosphorylates the GPCR operates at saturation and is less active than the G-protein Receptor Kinase (GRK), then the model is analogous to integral control of bacterial chemotaxis receptors. Inhibition of active chemotaxis receptors by demethylation is analogous to inactivation of active GPCRs by phosphorylation. This is a consequence of the fact that GRKs only phosphorylate receptors
associated with active proteins. The rate of receptor phosphorylation is: dRP/dt = VP
max - VKmax(A)/(KK+A) (where A is
the concentration of activated receptors, KK is the KM of the GRK for activated receptors, VP
max is the maximal activity of the phosphatase and VKmax is the maximal
activity of the kinase, GRK ). Thus, the activity at steady state will be: Ast= KKVP
max/(VKmax-VP
max) This is the set point (y0 in the model above). y is defined as the difference between the activity at time t (y1) and the activity at steady state (y0). Thus, at steady state, y = 0.
Increased ligand binding acutely increases u and elevates y1 to a value above y0, giving
a transient positive value for y (resulting in PI3K activation). At steady state, (y = 0) the rate of phosphorylation and dephosphorylation are equal. If one assumes that GRK only acts on active receptors (whether or not ligand is bound) then the net rate of phosphorylation at any instantaneous time will be directly proportional to y (the transient excess in active receptors over the steady state value). When y = 0 phosphorylation and dephosphorylaiton cancel out.The fraction of phosphorylated receptors (x) at any time t is then determined by the number of receptors in the phosphorylated state at time zero, x0 (e.g. prior to the perturbation due to increased ligand binding) plus the number of receptors that get phosphorylated during the interval in which the system was perturbed. This latter term is the integral from the time at which the perturbation (e.g. ligand unbinding) occurred t=0 to time t of ydt.
So x(t) = x0 + ydt
Notice that y can be + or - depending on whether ligand decreases or increases.Thus dx/dt = y = k(u-x) - y0
At steady state, dx/dt=y=0 and y1=y0 Notice that since k and y0 are constants, an increase in u (rapid binding of ligand) is ultimately offset by a slow decrease in x so that at steady state k(u-x) = y0.
0
t
Kin
ase
Kin
ase
P-Tyr
Tyr-P
Kin
ase
Autopho-transphorylation of low activity monomeric protein kinases in the ligand-induced dimer stabilizes the active state of each monomer, allowing further transphosphorylation at sites that recruit signaling proteins.
SH
2PI3K
Tyr-P P-Tyr
Regulation of protein-Tyr kinases
Kin
ase
Kin
ase
SH2 containing phosphoTyr phosphatases (e.g. SHP2) are preferentially recruited to activated receptors and play a dual role of transmitting additional signals (Ras activation) and turning off receptors.
SH
2
SHP2
P-Tyr
Tyr-P
Tyr-P P-Tyr
Tyr 1158
Tyr 1162
Tyr 1163
ATPPocket
INSULIN RECEPTOR CATALTIC DOMAIN (INACTIVE)
Prior to stimulation, protein-Tyr kinases have floppy activation loops (region containing Tyr 1157, 1162 and 1163 of the insulin receptor). As a consequence the enzyme has a low probability of being in the active conformation (~1%). Despite this low activity, when brought in proximity with a another low activity Tyr kinase (due to growth factor binding), cross-phosphorylation of respective activation loops can occur. Phosphorylation of the residues on this loop stabilizes the active conformation of the protein giving a ~100 fold increase in activity.
Activated Insulin Receptor
Peptide substrate
Integral Control of Receptor Protein-Tyr Kinases
The preferential dephosphorylation of activated Protein-Tyr kinases by SH2-containing phosphatases provides a potential mechanism for integral control. In response to an acute elevation in the level of ligand, the receptor will be rapidly activated, but in the continuous presence of the ligand, the phosphatase will ultimately return the kinase to a steady state activity that is determined by the affinity of the phosphatase for the activated kinase, the Vmax of the phosphatase and the Vmax of the kinase for transphosphorylation. Analogous to the set point for bacterial chemotaxis receptors one can show that:
Ast = KM-SHP2VKinmax/(VSHP2
max - VKinmax)
This simplified system does not reset to the same steady state as prior to receptor stimulation since VKin
max is dependent on receptor ligation. Modeling predicts an overshoot followed by return to a steady state that depends on ligand occupation. This is in agreement with observations at intermediate times (0 to 30 min.) following PDGF stimulation Exclusive ubiquitinylation, of activated protein-Tyr kinases (due to SH2-containing E3 ligases (e.g cbl)), leads to receptor internalization, providing a second mechanism of longer term shut-off that also models as integral feedback control.
Integral Control of PI3K
PI3K, when activated, phosphorylates lipids at a high rate but also autophosphorylates (on regulatory and catalytic subunits) at a slow rate, leading to inactivation.Assuming that the phosphatase that dephosphorylates PI3K is saturated by substrate, this could also lead to integral control of this enzyme.
Ras-GDP Ras-GTP
GEF (SOS)
GDP/GTP Exchange Factor (GEF) activate: analogous to GPCR
Effector
GAPGTPase Activating Protein Analogous to RGS
Effectors such as Raf (Ser/Thr kinase) or PI3K bind to activated Ras
Parallels between low molecular weight G protein (Ras, Rac Rho) regulation and heterotrimeric G protein regulation
basal slow
Heterotrimeric and low molecular weight GTP binding proteins have been retained and expanded during evolution because they have unstable activated states and can
spontaneously return to inactive states. Inactivation can also be accelerated by GAPs.
Signal Transduction in Eukaryotic cells is usually initiated by recruitment of signaling proteins to the plasma membrane. We have discussed three major mechanisms for acute and reversible protein relocation in response to cell stimulation. These mechanisms have the potential to amplify small signals.More importantly, recruiting signaling proteins from a 3-dimensional space (cytosol) to a 2-dimensional space (membrane) provides a mechanism for facilitating unfavorable multimeric interactions.
Activation of GTP-bindingproteins
Protein phosphorylation to create docking site
Generation of lipidsecond messengers
PI3K
GDP-Ras
SH2RasGEF
PI-4,5-P2
AKT
PH
PI-3,4,5-P3
Membrane
GTP-Ras
Membrane
Tyr P-Tyr
Membrane
Large AmplificationStochiometric Small Amplification
R28
1
34
5
1
3
Plasma MembraneSarraste
The PH domain of BTK binds the head group of PI-3,4,5-P3 and crystallizes as a dimer with the two binding pockets on the same surface. However, in solution, it behaves as a monomer.
Moving signaling proteins from the three dimensional environment of the cytosol to the two dimensional environment of the plasma membrane decreases the entropy difference between a monomeric and dimeric state.
Many signaling proteins may have evolved very weak free energies of homo or hetero-dimerization to insure that dimerization only occurs when confined on a two dimensional surface.
Membrane
Monomers
Dimers
Signal
Pólya's Random Walk Constants http://mathworld.wolfram.com/PolyasRandomWalkConstants.html
Let p(d) be the probability that a random walk on a d-D lattice returns to the origin. Pólya (1921) proved that
(1) p(1) = 1; p(2) = 1
but(2) p(d) < 1
for d > 2. Watson (1939), McCrea and Whipple (1940), Domb (1954), and Glasser and Zucker (1977) showed that
(3) p(3) = 1 - 1/u(3) = 0.340537….
where u(3) = 3/(2)3
_____dxdydz______3-cosx-cosy-cosz
Finch, S. R. "Pólya's Random Walk Constant." §5.9 in Mathematical Constants. Cambridge, England: Cambridge University Press, pp. 322-331, 2003.
Domb, C. "On Multiple Returns in the Random-Walk Problem." Proc. Cambridge Philos. Soc. 50, 586-591, 1954.
Glasser, M. L. and Zucker, I. J. "Extended Watson Integrals for the Cubic Lattices." Proc. Nat. Acad. Sci. U.S.A. 74, 1800-1801, 1977.
McCrea, W. H. and Whipple, F. J. W. "Random Paths in Two and Three Dimensions." Proc. Roy. Soc. Edinburgh 60, 281-298, 1940.
Montroll, E. W. "Random Walks in Multidimensional Spaces, Especially on Periodic Lattices." J. SIAM 4, 241-260, 1956.
Sloane, N. J. A. Sequences A086230, A086231, A086232, A086233, A086234, A086235, and A086236 in "The On-Line Encyclopedia of Integer Sequences." http://www.research.att.com/~njas/sequences/.
Watson, G. N. "Three Triple Integrals." Quart. J. Math., Oxford Ser. 2 10, 266-276, 1939.
Eric W. Weisstein. "Pólya's Random Walk Constants." From MathWorld--A Wolfram Web Resource. http://mathworld.wolfram.com/PolyasRandomWalkConstants.html
http://www.rwc.uc.edu/koehler/biophys.2ed/java/walker.html
http://www.krellinst.org/UCES/archive/modules/monte/node4.html
Further websites for random walks