Regulation of cell function by intracellular signaling Objectives: Regulation principle Allosteric and covalent mechanisms, Popular second messengers, Protein kinases, Kinase cascade and interaction. regulation 1 Regulation Principle regulation 2 1
Glycogenolysis Glycogenesis regulation 3 Activation of adenylyl cyclase and production of camp regulation 4 2
Activation of camp-dependent protein kinase or protein kinase A (PKA) regulation 5 Protein modulation can be achieved by binding to a regulator or adding a chemical group. regulation 6 3
Allosteric modulation Referring to the occupancy of protein binding sites by specific ligands resulting in changes in protein activity Dependent on the concentration of modulatory ligands Preferential for achieving a constancy of physiologic conditions, i.e. homeostasis regulation 7 Activation of camp-dependent protein kinase or protein kinase A (PKA) by cyclic AMP. (a) At low concentrations of camp, the enzyme exists as an inactive tetramer composed of two regulatory (R) and two catalytic (C) subunits. The tetrameric protein is inactive because the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. Binding of camp to the regulatory subunits causes release of the active monomeric catalytic subunits. (b) Structure of camp. regulation This unusual nucleotide, which acts as a "second messenger" in many intracellular signaling 8 pathways, controls the activity of many proteins. 4
Covalent modulation Referring mostly to protein phosphorylation, in which phosphate groups are added to a protein leading to changes in protein conformation and activity, Ideal for switching from one state to another, e.g., gene expression, enzymatic activation, control of ion channel activity. regulation 9 Phosphorylation and dephosphorylation regulation 10 5
Phosphorylation occurs in only three amino acids in high eukaryotes: serine, threonine and tyrosine, and the phosphate group comes from ATP. regulation 11 With a phosphate group, the local protein domain changes its interaction with other protein domains, leading to changes in protein structure and function regulation 12 6
Characteristics of covalent modulation Phosphorylation can be switched on (phosphorylation) and off (dephosphorylation). Phosphorylation is mediated by protein kinases. Dephosphorylation is mediated by phosphatases. A subtle balance between phosphorylation and dephosphorylation enables a control of gene expression, enzyme function, channel activity and other cell physiologic conditions. Specificity is determined by specific kinases and phosphatases in the dedicated signaling pathway. Amplification -- Activation of a kinase usually triggers a chain reaction in the cell. Each substrate in the cascades can become an enzyme to catalyze another reaction. regulation 13 Regulators Second Messengers Metabolic Substrates and Products Protein Kinases Protein Phosphatases regulation 14 7
Popular second messengers regulation 15 regulation 16 8
Activation of adenylyl cyclase by G s regulation 17 1) camp Hormone or transmitter Receptor G protein Adenylate Cyclase ATP camp AMP PKA Phosphodiesterase Effectors regulation 18 9
cgmp Hormone or transmitter Receptor G protein Quanylate Cyclase GTP cgmp GMP PKG Phosphodiesterase Effectors regulation 19 3) DAG/IP3 Ligand receptor G protein PLC DAG diacylglycerol IP3 inositol 1,4,5-triphosphate PIP2 phosphatidylinositol 4,5-bisphosphate PLC phospholipase C PIP2 DAG PKC Effector proteins IP3 Ca ++ (released from intracellular store) regulation 20 10
Figure 3-37. Specificity of cleavage of phospholipids by phospholipases A1, A2, C, and D. Susceptible bonds are shown in red. R denotes the polar group attached to the phosphate, such as choline in phosphatidylcholine (see Figure 5-27a) or inositol in phosphatidylinositol. (From Molecular Cell Biology ) regulation 21 Ca 2+ mobilization by IP3 Ca 2+ is pumped from the cytosol into the endoplasmic reticulum, which therefore serves as an intracellular Ca 2+ store. IP3 binds to receptors that are ligand-gated Ca 2+ channels in the endoplasmic reticulum membrane, thereby allowing the efflux of Ca 2+ to the cytosol. regulation 22 11
Function of calmodulin Calmodulin is a dumbbell-shaped protein with four Ca 2+ -binding sites. The active Ca 2+ /calmodulin complex binds to a variety of target proteins, including Ca 2+ /calmodulindependent protein kinases. regulation 23 Important Second Messenger Systems camp/cgmp systems. Phospholipidase C, diacylglycerol (DAG), inositol triphosphate (IP3), phosphatidylinositol 4,5 diphosphate (PIP2), and arachidonic acid systems. Ca ++, calmoduline. regulation 24 12
Cells can respond to extracellular signals carried by hormones and transmitters by changing cellular electrical, mechanical, metabolic, secretory and genetical activities. All of these changes start with a change in concentration of certain intracellular signal molecules that refer to the second messengers. The extracellular signal molecules thus refer to the first messengers. regulation 25 Protein Kinases regulation 26 13
Kinases can be generally classified: Serine/threonine kinases that phosphorylate specifically serine/threonine residues, and Tyrosine kinases that phosphorylate tyrosine residues, and Histidine kinases. regulation 27 A good number of genes are encoded for kinases. regulation 28 14
Most frequently, these kinases are divided into 4 categories: 1. The AGC group that includes the cyclic nucleotide-dependent protein kinase family such as PKA and PKG, the protein kinase C (PKC) family, the -adrenergic receptor kinase ( ARK) family, the ribosomal protein S6 kinase family, etc., 2. The CaMK group that includes the family of kinases regulated by Ca 2+ /calmodulin, the Snf1/AMPK family, etc., 3. The CMGC group that includes the cyclin-dependent kinase family, ERK/MAP kinase family, GSK3 family, casein kinase II (CK2) family, Clk family, etc., and 4. The conventional protein tyrosine kinase (PTK) group that includes near 100 receptor and non-receptor protein kinases. The last group is labelled as conventional to distinguish it from the dual-specificity kinases that are capable of phosphorylating tyrosine and serine/threonine residues, such as Clk, Mek/Ste7, Esk, Mik1, etc. Histidine kinase (HK) group, found in bacteria and yeast, responsible for adaptive responses to environmental stimuli, and often called twocomponent signal transduction system or phosphorylation relay system. regulation 29 Kinase Cascades Positive feedback control. Negative feedback control that turns off reaction or stabilizes the reaction at a constant level. regulation 30 15
Amplification regulation 31 Achieving steady state with negative feedback regulation 32 16
Positive and negative feedbacks regulation 33 Regulation of Membrane Proteins by Kinases regulation 34 17
regulation 35 Association of downstream signaling molecules with receptor protein-tyrosine kinases SH2 domains bind to specific phosphotyrosine-containing peptides of the activated receptors. regulation 36 18
Activation of phospholipase C by protein-tyrosine kinases Phospholipase C-g (PLC-g) binds to activated receptor protein-tyrosine kinases via its SH2 domains. Tyrosine phosphorylation increases PLC-g activity, stimulating the hydrolysis of PIP2. regulation 37 Ras activation downstream of receptor protein-tyrosine kinases A complex of Grb2 and the guanine nucleotide exchange factor Sos binds to a phosphotyrosine-containing sequence in the activated receptor via the Grb2 SH2 domain. This interaction recruits Sos to the plasma membrane, where it can stimulate Ras GDP/GTP exchange. The activated Ras-GTP complex then binds to the Raf regulation protein kinase. 38 19
Activation of the ERK MAP kinases Stimulation of growth factor receptors leads to activation of the small GTP-binding protein Ras, which interacts with the Raf protein kinase. Raf phosphorylates and activates MEK, a dual-specificity protein kinase that activates ERK by phosphorylation on both threonine and tyrosine residues (Thr-183 and Tyr-185). ERK then phosphorylates a variety of nuclear regulation and cytoplasmic target proteins. 39 Induction of immediate-early genes by ERK Activated ERK translocates to the nucleus, where it phosphorylates the transcription factor Elk-1. Elk-1 binds to the serum response element (SRE) in a complex with serum response factor (SRF). Phosphorylation stimulates the activity of Elk-1 as a transcriptional activator, leading to immediateearly gene induction. regulation 40 20
Cyclic AMP-inducible gene expression The free catalytic subunit of protein kinase A translocates to the nucleus and phosphorylates the transcription factor CREB (CRE-binding protein), leading to expression of camp-inducible genes. regulation 41 Regulation of gene expression by NF- B regulation 42 21
Allosteric Regulation A Kir6.2+SUR1 (ph 7.4) 0 M 100 M 30 M 10 M 3 M 1 M 0 M [ATP] 0 100-100mV 200pA 500ms B Kir6.2+SUR1 (ph 6.8) 0 M 300 M 100 M 30 M 10 M 3 M 1 M 0 M [ATP] 150 0-150mV 400pA 500ms regulation 43 A Kir6.2+SUR1 (no ATP) ph 7.4 7.1 6.8 6.5 6.2 7.4 WS 0 100-100mV 400pA 200ms B Kir6.2+SUR1 (100 M ATP) ph 7.4 7.1 6.8 6.5 6.2 5.9 7.4 WS 20pA 200ms C Kir6.2+SUR1 (1mM ATP) ph 7.4 7.1 6.8 6.5 6.2 5.9 5.6 7.4 WS regulation 44 50pA 200ms 22
A Normalized I 1.0 0.8 0.6 0.4 0.2 Kir6.2 C36, no ATP Kir6.2 C36, 100 M ATP Kir6.2 C36, 1mM ATP Kir6.2+SUR1, no ATP Kir6.2+SUR1, 100 M ATP Kir6.2+SUR1, 1mM ATP 0.0 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 ph B Normalized I 1.0 0.8 0.6 0.4 0.2 Kir6.2 C36, ph 6.8 Kir6.2 C36, ph 7.4 Kir6.2+SUR1, ph 6.8 Kir6.2+SUR1, ph 7.4 0.0 0 10 0 10 1 10 2 10 3 regulation 45 ATP (log M) A Kir6.2-H175A+SUR1 (ph 7.4) 0 M 100 M 30 M 10 M 3 M 1 M 0 M [ATP] 0 100-100mV 40pA 300ms B Kir6.2-H175A+SUR1 (ph 6.8) 100pA 300ms regulation 46 23
A Kir6.2 C36-K185E, no ATP ph 7.4 7.1 6.8 6.5 6.2 7.4 WS 100 0-100mV 100pA 200ms B Kir6.2 C36-K185E, 1mM ATP 200pA 200ms C Kir6.2 C36-K185E, 10mM ATP regulation 47 50pA 200ms A Normalized I 1.0 0.8 0.6 0.4 0.2 Kir6.2-K185E+SUR1, 1mM ATP no ATP Kir6.2-K185E+SUR1, no ATP 1mM ATP Kir6.2-K185E+SUR1, Kir6.2 C36-K185E, 1mM ATP 10mM ATP Kir6.2 C36-K185E, no ATP no ATP Kir6.2 C36-K185E, no ATP 1mM ATP Kir6.2 C36-K185E, 10mM ATP B Normalized I 0.0 1.0 0.8 0.6 0.4 0.2 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 ph Kir6.2 C36-H175A, ph 6.8 ph 6.8 Kir6.2 C36-H175A, ph 7.4 ph 7.4 Kir6.2 C36-H175K, ph 6.8 ph 6.8 Kir6.2 C36-H175K, ph 7.4 ph 7.4 Kir6.2-H175K+SUR1, ph6.8 ph6.8 Kir6.2-H175K+SUR1, ph7.4 ph7.4 Kir6.2-H175A+SUR1, ph6.8 ph6.8 Kir6.2-H175A+SUR1, ph7.4 ph7.4 Kir6.2+SUR1, ph7.4 ph7.4 0.0 0 10 0 10 1 10 2 10 3 10 4 regulation ATP (log M) 48 24
Interaction of two regulation mechanisms regulation 49 A GIRK1/GIRK4+MOR Control 100 nm DAMGO 1 µa 200ms B Pre-PMA 100 nm DAMGO 200ms C 60 50 40 30 20 10 0 Control No Pre-PMA Pre-PMA regulation 50 % Change in I 1 µa 25
A Control 100µM SP 30nM PMA GIRK1/GIRK4+NK1 2 µa B GIRK1-S185A/GIRK4-S191A+NK1 0 100-160mV 200 ms 1 µa C GIRK1-S185D/GIRK4-S191D+NK1 200 ms 1 µa 200 ms D 10 % I Inhibition 0-10 -20-30 -40 GIRK1/ GIRK4+NK1 GIRK1-S185A/ GIRK4-S191A+NK1 GIRK1-S185D/ GIRK4-S191D+NK1 GIRK1/GIRK4 regulation 51 Outside PKC Inside Phosphotase K + Open Closed regulation 52 26