1. fibroblast chemotaxis: more about positive feedback loops

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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 Presentation

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  • 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.

  • Fibroblasts chemotax toward growth factors03 hrs8 hrs12 hrs21 hrsPDGF-stimulated wound healing in mouse embryo fibroblasts

  • PI3K p110 Family Members110a110b110d110gKinasePIKC2Rasbindingp85bindingClass IaClass Ib

  • 03 hrs8 hrs12 hrs21 hrs WT PI3K-Ia DeletionDeletion 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 PDGF01020304050607080PI3K Ia deletionMigrated Cells3h8h15h21hP85a-/-;p85b-/-Wild typeLy294003PI3K inhibitor

  • unstimulatedPI3K IadeletionPDGFPDGF + WMWild TypeDefect in PDGF-induced lamellipodia formationin MEFs defective in class Ia PI3KBrachmann et al., 2005 Mol Cell Biol 25, 2593

  • SH3Cdc42bindingSH2SH2MembraneCatalyticRasBindingGTPRasGTPCDC42P-TyrP-Tyr

    Class Ia PI 3-KinasePIKTyr KinaseC2Class Ia PI3K has multiple domains for signal input, allowing it to act as an AND GATE or possibly an OR GATEp85 regulatoryp110 catalytic

  • Class Ia PI3K mediates growth factor-dependent cortical actin formation TyrKinaseReceptorGrowthFactorP-TyrGTPRasRacGEF?PIP2

  • Wild Type MEFs PI3K Ia deleted 5 min PDGF [ng/ml]: 0 1 3 10 0 1 3 10Erk-PErkDeletion 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, 2593Thus, as in Dictyostelium, there appears to be a positive feedback loop between PI3K and Ras in fibroblasts.

  • Erk-PErk Control Double KO PDGF: - + - +Rac GTPRacp85aGST-CRIB pulldownReduced PDGF-induced Rac activationin MEFs lacking class Ia PI3KBrachmann et al., 2005 Mol Cell Biol. 25, 2593

  • Overexpression of a Rac GEF (Vav2) induces lamellipodia formationin MEFs lacking Class Ia PI3KBrachmann et al., 2005 Mol Cell Biol. 25, 2593

    Rhodamine-Phalloidin(Actin)

    Vav2Wild Type PI3K Ia deleted

  • PI3K is involved in both local Ras and local Rac positive feedback loops++RacGTPp85p110PIP3GEFPH?RasGTP?PDGFReceptor

  • Conclusions1. Growth Factor Receptors stimulate Class Ia PI3K through PhosphoTyr residues of receptors binding to SH2 domains, while GPCRs stimulate Class Ib PI3K through bg 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?

  • ReceptoraGDPbgGPCR ACTIVATION

  • ReceptoraGDPbgLigand-inducedConformationalChange
  • ReceptoraGTPbgSignal Termination, Downregulation and Reset to Basal StateEffector 1Effector 2

  • ReceptoraGTPbgSignal Termination, Downregulation and Reset to Basal StateEffector 1

  • ReceptoraGTPbgSignal Termination and Reset to Basal StateP P PArrestinEffector 1Only 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, 4649If 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 bg proteins. The rate of receptor phosphorylation is: dRP/dt = VPmax - VKmax(A)/(KK+A) (where A is the concentration of activated receptors, KK is the KM of the GRK for activated receptors, VPmax 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= KKVPmax/(VKmax-VPmax) 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) - y0At 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.

  • 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.Regulation of protein-Tyr kinases

  • 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.

  • 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 ReceptorPeptide 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/(VSHP2max - VKinmax)

    This simplified system does not reset to the same steady state as prior to receptor stimulation since VKinmax 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-GDPRas-GTPGEF (SOS)GDP/GTP Exchange Factor (GEF) activate: analogous to GPCREffectorGAPGTPase Activating Protein Analogous to RGSEffectors such as Raf (Ser/Thr kinase) or PI3K bind to activated RasParallels between low molecular weight G protein (Ras, Rac Rho) regulation and heterotrimeric G protein regulationbasalslowHeterotrimeric and low molecular weight GTP binding proteins have been retained and expanded during evolu

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