a As single footstep will not make a path on the earth, so a single thought will not make a pathway in the mind. To make a deep physical path, we walk again and again. To make a deep mental path, we must think over and over the kind of thoughts we wish to dominate our lives. Henry David Thoreau We think in generalities, but we live in detail. Alfred North Whitehead Of course I'm crazy, but that doesn't mean I'm wrong. Robert Anton Wilson
Announcements
Quiz 9 is due Wednesday 12/9, at noon Final is December 16, 8-10 AM in STEW 183 (Loeb) Final is cumulative, but 80% focused on material since exam 3 Review questions and guidelines to material to know from first three exams will be given out early next week
Second messengers
Activated by effector that has been activated by first messenger (ligand) or the transduction of the ligand binding, such as activation of a G-protein by a ligand-activated receptor. Examples include: cyclic AMP (cAMP) Ca++ Phosphoinositides, such as PIP2 Inositol trisphosphate (IP3) Diacylgycerol (DAG) Cyclic GMP (cGMP, mostly in vision) Nitric oxide
Sensitization of the gill withdrawal reflex reveals three phases of memory
Non-associative memory Light touch evokes a small withdrawal. A single shock will sensitize the gill withdrawal to a touch for about 1 hour 4 shock will sensitize the withdrawal for 1 day, and 4 sets of 4 shocks for 4 days will sensitize the withdrawal for weeks This gives 3 different time windows for sensitization that have distinct molecular mechanisms Kandel et al. 2001
Steps in short-term sensitization in Aplysia
Short term (hour): PKA directly phosphorylates K channels, leading to increased transmitter release Intermediate term (hours to 1 day). PKA activation of CREB ultimately results in ubiquitination of PKA Long-term (weeks). Multiple gene activation for addition of new synaptic release sites
Kandel et al. 2001
PKA anchoring proteins (AKAPs) ensure that PKA phosphorylates the proper substrates
The PKA binding domain is shared between different AKAPs, but different AKAPs have specific binding sites for the receptors that they are coupled to, forming a signaling hub In this example, AKAPs associated with channels, mitochondria, nucleus, cytoskeleton
Regulation of phosphorylation by kinases and phosphatases is at the heart of many signaling pathways
Shown is a general scheme of a cascade of kinase regulation steps that ultimately leads to activation of a transcription factor
Receptor tyrosine kinases
Trans-autophosphorylation means that one subunit will phosphorylate the other subunit in the dimer The phosphorylated receptors will now bind to target proteins with SH2 or PTB domains and activate them As a reminder, domains refer to conserved amino acid sequences with specific functions Growth factors such as epidermal growth factor and platelet derived growth factor use protein tyrosine kinases
Youve activated the receptor, now what? Different signaling proteins
a) b) c) d)
Adaptor proteins: link signaling proteins with SH2 domain Docking proteins: can add tyrosine phosphorylation sites Transcription factors: Activated factor goes directly to nucleus to regulate genes (STAT protein shown in example, involved in immune function) Other signaling enzymes, such as PLC-gamma, PI3K, phosphatase
MAP kinase pathway
1) 2) 3) 4) 5) Glucose regulation, cell proliferation and differentiation Dysregulation, especially via Ras, is a common route to cancer Receptor activation induces intracellular phosphorylation An adaptor protein such as Grb2 can bind to activated receptor and to Sos (a GEF) Sos activates Ras-GDP to Ras-GTP
Back to G-proteins again
Ras, shown to the left, is a class of small G-proteins involved in many processes, including the MAPK pathway Switch I and II are domains that change conformation when guanine is bound
MAP kinase pathway
Sos activates Ras-GDP to RasGTP Enter the kinase cascade, Raf, MEK, ERK MAPKKK is a MAP kinase kinase kinase, because it phophorylates sequentially two steps away from MAPK MEK is a dual-specificity kinase and can work on tyrosine or serine-threonine Activated MAPK into the nucleus to phosphorylate transcription factors Transcription factors regulate genes MAPK turned off by MAPK phosphatase (MKP-1) upregulated by transcription factor
Inhibitory
Glucose storage vs. mobilization: competing intracellular pathways
Synthesis
Breakdown
The same general MAPK path can be used for different purposes in different organism and tissue types Glucose regulation is used across many species Need glucose for energy Can store glucose for future use Glucose stored as glycogen Glucagon and epinephrine breakdown cause of glycogen to glucose by upregulating glycogen phosphorylase Insulin triggers the formation of glycogen from glucose by upregulating glycogen synthase
Dual PKA action activates glycogen breakdown and inhibits glycogen synthesis pathways
2 1
Phosphorylation by PKA activates phosphorylase kinase (1) and inhibits glycogen synthase by activating a kinase to phosphorylate glycogen synthase (2)
CREB/CRE are critical for coupling receptor activation to long-term changes in the cell
cAMP response element binding protein (CREB) is a common target for intracellular pathways (Aplysia sensitization, glucose regulation, etc) PKA phosphorylates CREB and CREB dimerizes CREB binds to the cAMP response element (CRE), which is a nuclear DNA sequence. CREB binding will upregulate transcription of multiple genes that are coupled to CRE, usually related to a unified function (e.g. growth, apoptosis (cell death), etc.)
Insulin reduces blood glucose and promotes glycogen synthesis
Insulin receptor consists of two alpha and two beta subunits Insulin receptor is a tyrosine kinase Insulin binding causes phosphorylation of beta subunits and insulin receptor substrates (IRSs)
After activation of the insulin receptor
IRS-1 is a docking protein with multiple tyrosine phosphorylation sites that binds to the activated insulin receptor through a PTB domain IRS-1 can bind to Grb2/Sos to activate Ras PI3K can also bind to IRS-1. PI3K makes PIP3 from PIP2
Further downstream of insulin binding
PI3K makes PIP3 PIP3 interacts with two enzymes through interactions with a PH domain PKB and PDK1 are serine threonine kinases PIP3 binding to PKB activates PKB and allows it to be phosphorylated by PDK1 A second kinase also phosphorylates PKB Phosphorylated PKB dissociates from the membrane and signals glucose uptake, glycogen synthesis, and protein synthesis
PKB is required to upregulate glucose transporter insertion into plasma membrane
Activated PKB enhances glucose transporter fusion, thereby increasing glucose uptake from blood into cells Activated PKB also phosphorylates glycogen synthase kinase 3 (GSK-3) which is the negative regulator of glycogen synthase GSK-3 is inactivated by phosphorylation by PKB, allowing glycogen synthase to operate at high rates
GSK3 and glycogen synthase are both inactivated by phosphorylation
GSK3, - reg.
PKA, - reg.
PKB phosphorylates GSK3 to inactivate it, so you have inhibition of an inhibitory regulator. Therefore, PKB indirectly permits glycogen synthase to operate.
Diabetes
There are 20.8 million children and adults in the United States, or 7% of the population, who have diabetes. (from American Diabetes Association) Type 1 diabetes: 5-10% of cases, insulin is not produced Type 2 diabetes: 90-95% of cases, either insufficient insulin or desensitized insulin response Elevated levels of insulin overstimulate target cells in the liver & elsewhere in the body, leading to a condition called insulin resistance (target cells stop responding to the presence of the hormone) Obesity and inactivity are major risk factors for Type 2 diabetes, which is also correlated with increased risk of heart disease or stroke
Calcium and PKB can regulate insertion of glucose transporters into membrane
Ca++ is required to secrete insulin from the beta pancreatic cells Ca++ binds to calmodulin and activates it. Calmodulin can then bind to Ca/calmodulin-dependent kinases (CaM kinases) In this example CaMKII promotes insulin release
Easom 1999
Calcium ions are also a key component of many signaling pathways
In this example, a cerebellar neuron has been filled with a fluorescent calciumsensitive dye (Fura-2) Calcium release from intracellular IP3 channels was induced by repetitive synaptic stimulation The level of intracellular Ca++ will determine the dynamic synaptic strength
Calcium sources for signaling
Ligand-gated receptors, such as the NMDA glutamate receptor Voltage-gated Ca++ channels IP3 intracellular channels Ryanodine (RyR) intracellular channels Calcium pump of the smooth endoplasmic reticulum (SERCA) Depicted are the calcium sources for a cardiac muscle cell Depolarization allows Ca in through channels Calcium induces calcium release from RyR channels Ca removed by SERCA and Na/Ca exchanger in plasma membrane
Signaling pathways are not independent and can interact
Convergence: Signals from multiple receptors activate a common effector, such as Ras or Raf Divergence: Signals produced by a single receptor activate more than one different effector, such as PKC and IP3 Crosstalk: Components of signaling pathways are shared between pathways, such as [cAMP], PKC, ATP