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POM UNC > Cell Signaling > Flashcards

Cell Signaling Flashcards

1
Q

Draw the G-protein signaling cascade

A

ligand binds to GPCR, set of alpha helices open up, GEF domain opens up, G-protein’s alpha subunit is activated (GTP bound), dissociates from beta/gamma subunits, activates effector enzyme, effector enzyme releases secondary messengers, which activate protein kinase, which changes gene expression

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2
Q

Draw Gs-protein signaling cascade

A

hormone binds GPCR. GPCR opens up, GEF activates G protein alpha subunit. alpha subunit leaves and binds to adenylate cyclase, which converts ATP to cAMP. cAMP activates Protein Kinase A (PKA) by removing regulatory subunits, and PKA goes on to impact target gene

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3
Q

Draw Gq-protein signaling cascade

A

Hormone binds GPCR. GPCR opens up. GEF activates G protein alpha subunit. Alpha subunit leaves and binds to phospholipase Cbeta, which converts PIP2 to DAG and IP3. IP3 interacts with endoplasmic reticulum to induce a release of calcium ions, which activate Protein Kinase C (PKC). PKC does its thing

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4
Q

Draw Gi-protein signaling cascade

A

Hormone binds GPCR. GPCR’s stabilizing salt bridge breaks apart, the GEF domain opens up, G protein is GTPed. Alpha unit goes to inhibit the adenylate cyclase pathway, stopping any PKA formation. The beta/gamma subunit goes to activate phospholipase Cbeta, which transforms PIP2 into DAG and IP3, which goes to release calcium from the endoplasmic reticulum and ultimately induce gene expression.

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5
Q

What is the Ras pathway?

A

Ras is probably the most common (or most talked about) monomeric G protein. Ras is activated by Receptor Tyrosine Kinase or Cytokine Receptor. Receptor cross phosphorylates. Adapter protein’s SH2 region binds to the phosphorylated tyrosine residues on the receptor, and uses its SH3 domain to bind to a polyproline-GEF, which has a PH domain (stabilizes by bonding with membrane). Ras is activated by the GEF causing it to drop GDP and pick up GTP. It goes on to activate Raf, which activates MAPK, which activates MAPKK, which activates MAPKKK, which affects gene.

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6
Q

What is a receptor tyrosine kinase? Draw its mode of action on the cell.

A

A receptor tyrosine kinase is enzyme receptor which phosphorylates tyrosine residues after binding with a ligand. RTKs bind ligands and then form a dimer with another RTK. The dimer crossphosphorylates (autophosphorylates) and the phosphorylated tyrosine residues are used to activate other effector enzymes. Because RTK is a dimer, it can act through two pathways at the same time, achieving slightly different results towards the same goal. RTK can act through the g protein (Ras) pathway, or through the effector enzyme (PI3K) pathway.

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7
Q

What is the PI3K pathway? Draw

A

A pathway used by cytokine receptors and receptor tyrosine kinases. PI3K has two subunits, a regulatory subunit and a catalytic subunit. PI3 Kinase is activated by phosphates on RTK, and converts ATP–> ADP, adding the phosphate group to PIP2 (a molecule found in the cell membrane). PIP3 is a secondary messenger used to attract Protein Kinase B (PKB) and a kinase that activates PKB to the cell membrane. PKB is activated and affects gene expression.

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8
Q

What are GEFs?

A

Guanine Nucleotide exchange factor - increases the ability of GDP to pop off (dissociate) from G-protein

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9
Q

What are GDIs?

A

Guanine Nucleotide Dissociation Inhibitors - decreases the ability of GDP to dissociate from G-protein

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10
Q

What are GAPs?

A

GTPase activating protein - GTPase turns GTP to GDP, so GAP increases the kcat-GTP (speed/quantity of GTP turning to GDP)

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11
Q

What are GIPs?

A

GTPase inhibiting proteins - GTPase turns GTP to GDP, so GIPs decrease kcat-GTP

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12
Q

If you want to stop gene expression promotion by targeting G-protein, what would you do?

A

Increase Kcat-GTP, decrease Kd-GDP. You want G-protein to be stuck with GDP bound. Adding GDIs and GAPs would increase hydrolysis from GTP–> GDP and decrease speed at which or even amount of GDP that drops off of G-protein

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13
Q

How are g-protein subunits kept near the membrane?

A

They’re locked into the membrane through fatty acid “greasy groups” which, being hydrophobic, are stable while anchored in the membrane.

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14
Q

What does GPCR look like?

A

Seven alpha helices, wrapped up like a burrito. When it is activated a salt bridge between glutamic acid on one alpha helix and lysine on another alpha helix is broken, and the GPCR opens up, exposing a G protein binding surface. The GPCR has 3 intracellular loops connecting the alpha helices, and 3 extracellular loops. The N terminus is in the extracellular region. The C terminus is in the intracellular region

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15
Q

Why are RTKs and G protein Receptors linked with cancer?

A

When a mutation allows RTKs or GPCRs to be active without a bound hormone, the cell can be pushed to relentlessly multiply, a hallmark of cancer.

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16
Q

What is positive feedback?

A

As a product is made, its increasing concentration encourages further production.

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17
Q

What are regulatory subunits? Regulatory domains?

A

A regulatory subunit is an isolated protein which interacts with an enzyme/kinase and either inhibits or activates the catalytic cleft. The catalytic cleft is where phosphorylation (and other) reactions take place. Regulatory domains are similar, but are attached (part of the same peptide chain) to the enzyme.

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18
Q

What is negative feedback?

A

As a product is made, its increasing concentration discourages further production. Most products have some form of negative feedback with their pathways.

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19
Q

What is a catalytic cleft?

A

The location on a protein kinase where ATP or GTP and the target substrate are close enough to undergo a reaction and transfer a phosphate to the substrate.

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20
Q

How does the regulatory subunit/domain block kinase activity?

A

The regulatory subunit/domain literally blocks access to the catalytic cleft, keeping the substrate from binding and being phosphorylated.

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21
Q

What does adenlyate cyclase do?

A

It turns ATP into cAMP

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22
Q

What does phospholipase Cbeta do?

A

It turns PIP2 into DAG and IP3. IP3 goes on to release calcium ions from the ER.

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23
Q

What does guanylate cyclase do?

A

It turns GTP into cGMP

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24
Q

How do steroids effect gene expression?

A

Steroids are able to sneak through the hydrophobic plasma membrane and the nuclear envelope to directly induce change of gene expression. Steroids will bind to an intracellular receptor, which directly binds to DNA and induces a change in transcription.

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25
Q

How do steroids effect gene expression?

A

Steroids are able to sneak through the hydrophobic plasma membrane and the nuclear envelope to directly induce change of gene expression. Steroids will bind to an intracellular receptor, which directly binds to DNA and induces a change in transcription.

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26
Q

What is a cytokine receptor?

A

A cytokine receptor is kind of like a receptor tyrosine kinase, except it doesn’t have inherent enzymatic activity. Instead it has spots for JAK (janus kinases) to bind. These kinases then phosphorylate each other and activate the signaling pathways. Cytokine receptors can act through effector enzymes (PI3K) g proteins (ras) or STATs (this is a super efficient way to directly engage the genetic material). Cytokine receptors are transmembrane proteins

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27
Q

Draw the STAT pathway for a cytokine receptor

A

Cytokine binds to CR subunit. CR combines. JAK binds. JAK autophosphorylates. JAK phosphorylates STAT. STAT forms dimers. STAT binds to DNA and causes transcription.

28
Q

Are cytokine receptors present as dimers or monomers?

A

Cytokine receptors have two subunits which come together before becoming active and leading to a signal cascade. One side usually has the JAK activation domain and another has the cytokine receptor.

29
Q

What are the different domains in a kinase?

A 
ATP binding lobe (with phosphate anchor)
Substrate binding lobe
Catalytic cleft (in between lobes)
Regulatory domain/subunit.
Activating loop
30
Q

What are the different ways adding phosphates can regulate kinase activity?

A

Adding phosphates to the phosphate anchor in the ATP binding lobe will inhibit the Kinase.
Adding phosphates to the activating loop (attached to the substrate binding lobe) will activate the kinase.

31
Q

What are the ways taking phosphates away can regulate kinase activity?

A

Taking phosphates away from phosphate anchor will allow kinase to operate (not really activate, but un-deactivate)
Taking phosphates away from activating loop will deactivate the kinase.

32
Q

How do secondary messengers get protein kinases to activate?

A

Secondary messengers can bind to regulatory subunit/domain and pull the domain away from the catalytic cleft, opening it up for enzymatic activity. OR secondary messengers can activate the regulatory domain, getting it to interrupt the catalytic cleft

33
Q

What is the most common form of kinase-based regulation of kinases?

A

Kinase will add phosphates to the activation loop on the protein kinase targeted, protein kinase will become active.

34
Q

What are the two types of signal hormones?

A

Lipophilic (hydrophobic) - sneak through membranes and act directly on nucleus
Hydrophilic - have to operate through receptor enzymes, cytokine receptors, GPCRs.

35
Q

What are the different families of proteins that impact G-protein activity?

A

G proteins want to be bound to GTP to be active. Anything that takes GTP away from G protein will push G protein towards inactivity.

Guanine nucleotide dissociation inhibitors (GDIs) keep GDP bound to G protein
Guanine nucleotide exchange factors (GEFs) pull GDP off G protein
GTPase activator proteins (GAPs) encourage GTP–> GDP hydrolysis
GTPase inhibitor proteins (GIPs) decrease GTP–> GDP hydrolysis

36
Q

Rules of thumb for positive/negative regulation?

A

Positive regulatory interactions favor the active conformation
Negative regulatory interactions disfavor the active conformation (secondary messengers often undo the negative regulation)

37
Q

What are secondary messengers?

A

Come out of effector enzymes, activate protein kinases, which impact gene expression of target gene

38
Q

What is the GPCR salt bridge?

A

An ionic interaction between a glutamic acid on one alpha helix of the receptor and a lysine on another alpha helix of the receptor. Keeps the complex tightly packed. Breaks in the presence of hormone.

39
Q

What does phospholipase Cbeta do?

A

It converts PIP2 to DAG with a biproduct of IP3, which is used to activate calcium ion release from the endoplasmic reticulum (there’s an IP3 receptor on the ER)

40
Q

How does calcium ion impact regulation of Protein Kinase C?

A

Calcium (when paired with DAG) bind to PKC’s regulatory domain and weaken the interaction between the domain and the catalytic cleft. This allows ATPs and substrates to bind and interact

41
Q

Are Janus Kinases natively active or inhibited?

A

JAKs are naturally inhibited by a regulatory domain. When they bind to cytokine receptors the regulatory domain is pushed away and JAK becomes active.

42
Q

What does adenylate cyclase do?

A

It takes ATP and converts it to cAMP, which is used to activate Protein Kinase A by “distracting” the regulatory subunit and opening the kinase up for activity.

43
Q

How many alpha helices does a GPCR contain?

A

7!

44
Q

What is a heterotrimeric G protein?

A

A g protein with three subunits (alpha, beta, gamma). Alpha is what binds to GTP. beta and gamma are always together. They don’t do much except in G(i) proteins. In G(i) proteins the activated alpha subunit inhibits adenylate cyclase, but the beta/gamma subunit activates phospholipase Cbeta, and encourages PKC activation.

45
Q

How does cAMP impact PKA?

A

cAMP reverses the “fake substrate” psuedosubstrate’s strength of binding with the catalytic cleft. The psuedosubstrate leaves and actual substrate is able to come in and be phosphorylated. It breaks up the regulator/kinase complex (2RK) into (2R+cAMP) + 2K

46
Q

What are the different types of interaction domains found in signaling proteins?

A

PH - interacts with cell membrane
SH2 - interacts with tyrosine phosphate heads
SH3 - interacts with polyproline
PTB - interacts with polytyrosine or tyrosine phosphate

47
Q

How are signaling proteins’ catalytic domains organized?

A

They often have a modular collection of interaction domains to bind with multiple molecules at the same time or add versatility to the protein’s function. Interaction domains are independently translated ~100 amino acid long peptide chains that are used to stabilize/facilitate interactions

48
Q

How are receptor tyrosine kinases able to activate multiple signaling cascades at once?

A

RTKs have two monomers that form a dimer. Each monomer has multiple phosphorylation sites, and is able to activate an independent signaling cascade. These cascades are often used in parallel to affect greater changes in the cell.

49
Q

What are the structural components of an RTK?

A

Two halves. Each half is identical. Extracellular ligand/hormone binding domain. Transmembrane domain. Intracellular tyrosine kinase domain.

50
Q

What is dimerization?

A

In cytokine receptors or RTKs two units need to combine before activating effector enzymes or causing any changes in cell behavior.

51
Q

What is the Ras signaling pathway overview?

A

Ras –> Raf –> MAPK –> MAPKK –> gene (or MAPKKK)

52
Q

What is the PI3K signaling pathway

A

PI3K –> PIP2 to PIP3, PIP3 –> PKB + PKBK –> activated PKB

53
Q

What classes of proteins typically regulate activation states of intracellular signal relay components in response to a hydrophilic hormone binding its cell-surface receptor?

A

G proteins, ion channels, receptor protein kinases

54
Q

What relationship of the key equilibrium and catalytic rate constants govern the amount of G-protein in the active state?

A

Kd-GDP (kick GDP off G protein)
Kcat-GTP (convert GTP to GDP)
there isn’t really a constant for empty G protein binding to GTP because we assume it happens super fast
Mathematical relationship can be approximated as KdGDP/KcatGTP = Active/Inactive

55
Q

What classes of regulatory proteins function to increase the fraction of G-protein in the active state?

A

GTPase inhibitors stop GTP from turning to GDP

Guanine nucleotide exchange factors help pull GDP off the G protein

56
Q

What step in the G-protein cycle do GIps, GAPs, GEFs, and GDIs work?

A

GIP and GAP are when GTP is bound (G protein is active)

GEF and GDI are when GDP is bound (G protein is inactive)

57
Q

What mechanisms are commonly used to inhibit activity of protein kinases in the cell? Which is relieved by the action of second messengers?

A

Inhibit G protein activation. Inhibit adenlyate cylase activation. Use regulatory subunit or domain to keep enzyme “stuffed” and unable to bind substrates. Second messengers often act on regulatory subunits/domains.

58
Q

What are the structural characteristics shared by all G protein coupled receptors?

A

Seven alpha helices organized in a sort of shell. Tightly packed until hormone binds, and then this salt bridge that was holding them together is dissolved and the helix complex opens up a bit, allowing for enzymatic factors to bind. Also 3 intracellular and 3 extracellular loops. C terminal is intracellular, N terminal is extracellular.

59
Q

Glucagon binding to its receptor activates a Gs family member. What protein kinase activity is induced by Glucagon? What mechanism activates this kinase?

A

Gs = adenylate cyclase activates cAMP which activates PKA by releasing the regulatory subunits.

60
Q

Angiotensin binding to its receptor activates a Gq family member. What second messenger molecules are increased in the cell as a result of angiotensin binding to its receptor?

A

IP3, DAG, ultimately Ca2+

61
Q

Which interaction domain mediates binding to phosphotyrosine residues?

A

SH2, PTB

62
Q

Which interaction domain mediates binding to membrane lipids?

A

PH

63
Q

Compare G-protein cycles characteristic of heterotrimeric G proteins and monomeric Ras.

A

Similar, except alpha subunit has to dissociate from the beta/gamma subunit. And G proteins (not Ras) don’t have to deal with the adapter protein.

64
Q

How do activated RTKs promote activation of PKB?

A

RTKs activate PI3K pathway, which causes PKB to become present.

65
Q

What is the signal relay that is specific to Cytokine Receptor mediated signaling?

A

STAT signaling

POM UNC (21 decks)

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