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Biochem II Self Study > Lecture 20 (exam 3) > Flashcards

Lecture 20 (exam 3) Flashcards

1
Q

Enzyme coupled receptors

A

are either enzymes or are very closely associated with enzymes

  • Many receptors are transmembrane proteins with single membrane-spanning domain.
    This is particularly true for those that regulate growth.
  • Substrate binding causes dimerization
  • Receptor dimers are enzymatically active (often with protein kinase activity)
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2
Q

Enzyme coupled growth factor receptors

A

are structurally and functionally different from G protein linked receptors

most in this class are protein kinases, some are protein phosphatases or guanylyl cyclase activity

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3
Q

Enzyme coupled receptors examples

A
  • Receptor tyrosine kinases (RTKs)
  • Tyrosine kinase associated receptors
  • Receptor serine/threonine kinases
  • receptor guanylyl cyclases
  • receptor tyrosine phosphatases
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4
Q

Receptor tyrosine kinases (RTKs)

A

phosphorylate on specific tyrosines (growth factor receptors)

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5
Q

Tyrosine kinase-associated receptors

A

non-covalent association with intracellular tyrosine kinases (cytokine receptors)

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6
Q

Receptor serine/threonine kinases

A

phosphorylate specific serines or threonines ((TGFβ receptors)

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7
Q

Receptor guanylyl cyclases

A

synthesize cGMP

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8
Q

Receptor tyrosine phosphatases

A

remove phosphate from tyrosines

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9
Q

RTKs are…

A

the most abundant type of enzyme-coupled receptor, with about 60 members classified into 20 structural families

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10
Q

RTKs regulate

A

cell proliferation, cell growth, cell differentiation, cell migration, and during during development, cell fate

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11
Q

Growth factors

A

a class of signaling molecules

RTKs were discovered by studying GFs

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12
Q

Mechanisms of Dimerization for RTKs

A
  1. The ligand can be a dimer itself and thus bind to the two receptors simultaneously (as above).
  2. A monomeric ligand can bind to 2 receptors simultaneously to bring them together.
  3. Two ligands can bind independently to 2 receptors to bring them together.
  4. Some receptors like the insulin receptor are already dimers, and the ligand just induces a conformational change that activates the kinase activity.
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13
Q

RTKs Contain an Intracellular Tyrosine Kinase Domain

A
  • In the inactive or basal state, RTKs are monomers anchored in
    the plasma membrane by a single transmembrane domain
  • The intracellular domain of RTKs contains a ligand-activated
    tyrosine kinase domain
  • The receptors themselves are the initial substrates of that
    tyrosine kinase activity
  • Binding of ligand causes receptor monomers to dimerize in the
    the plasma membrane, which activates the tyrosine kinase activity
    of one or both monomers
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14
Q

Dimerization of RTKs

A
  1. Trans phosphorylation = autophosphorylation:
    the activated tyr kinase domains phosphorylate
    each other (‘standard’ mechanism)
  2. Conformational changes in
    the domains in response to
    ligand binding ends up having
    one tyr kinase domain
    (activator) activating the other
    the Tyr kinase domain
    (receiver), and the receiver
    then phosphorylates both
    subunits (EGF-R mechanism)
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15
Q

Once the Internal Domains of the RTK Are Phosphorylated, They Serve as

A

Docking sites for signaling proteins

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16
Q

Binding of phosphotyrosine

A

SH2 Domain and PTB domain

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17
Q

Binding of Phosphotyrosine: SH2 Domains

A

The first modular interaction domain was shown to depend on the PTM of the substrate

  • ~100 amino acids in length
  • Central β-sheet separates two α-helices
  • One binding site on each side of βsheet:
    • Positive ‘phosphotyrosine pocket’
    • Variable ‘specificity pocket’
  • Each binding pocket contributes roughly half of the binding energy
  • Phosphorylation increases affinity
    1000x
  • Bidentate ionic interaction with ArgβB5
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18
Q

Binding of Phosphotyrosine: PTB Domains

A

Two orthogonal β-sheets form a
sandwich, capped by a single Cterminal α-helix

  • Binds preferentially to “NPxpY”
    motif, where x can be any amino
    acid and pY is the phosphotyrosine
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19
Q

‘Adaptors’ link-activated RTKs or other

A

signaling pathways

  • adaptors have binding domains that scaffold multiple proteins, bringing them in close proximity
  • adaptors have no enzymatic activity
20
Q

Grb2

A

is an adaptor that binds RTKs

links the MAP kinase cascade to RTKs in some cases

is composed of little more than a central SH2 domain

21
Q

SH3 domains bind

A

proline-rich regions

contain two binding grooves, each binds one proline and one other (hydrophobic) amino acid

polyproline type II (PPII) helices

22
Q

Polyproline type II (PPII) helices

A

are common recognition motifs (PxxP sequences)
- rigid, left-handed helix
- three residues per turn
- pseudo symmetry allows N-C or C-N binding

23
Q

RTKs are proto-oncogenes and often become…

A

oncogenic

The RTK can become mutated so that it dimerizes and becomes
constitutively active without the ligand, or the external domain can be truncated, which also activates the receptor without a ligand.

Anything that results in a constitutively active kinase domain is bad news

24
Q

Mutation of the Her2 and EGF Receptors Leads to

A

activation without ligand

both receptors bind EGF

Her2/Neu is often mutated or overexpressed in ‘hormone-independent’ breast cancer (BCa)

25
Q

Major pathways activated by RTKs

A

cell proliferation, cell growth, cell survival

26
Q

RTK Pathways (II): Phospholipase C-y

A
  • Phospholipase C-γ functions the same as PLC-β: cleavage of PIP2 to make two secondary messengers (DAG and IP3).
  • This allows RTKs to increase cytoplasmic Ca2+ concentration
    and activate Protein Kinase C
27
Q

Compare/contrast PLC-β and PLC-γ

A
  • Major differences are in binding domains in the c-terminus
  • PLC-γ has several Tyrosine residues that are phosphorylated
    by the activated RTK
28
Q

How do you know if something has been phosphorylated?

A
  1. Western blot with phosphotyrosinespecific antibody (or anti-phosphoserine, etc. antibody)
  2. Radio-labeled phosphate incorporation
  3. Tandem mass spectroscopy (MS/MS)
    Electron Transfer Dissociation (ETD)
29
Q
  1. Tandem mass spectroscopy (MS/MS)
    Electron Transfer Dissociation (ETD)
A

is a gentle fragmentation method that does not cleave off the phosphates, and allows the
precise identification of phosphorylated residues.

30
Q

MAP kinase cascade

A

Mitogen activated protein kinase

31
Q

mitogens

A

signaling molecules that induce cell proliferation such as growth factors, estrogen and testosterone by triggering mitosis

32
Q

Signaling from RTK to Ras

A

Grb2 recognizes a specific phosphorylated tyrosine on the RTK by means of an SH2 domain and recruits Sos by means of two SH3 domains. Sos stimulates guanine nucleotide exchange, which activates Ras

33
Q

Grb2

A

is just an adaptor protein, composed of SH2 and SH3 domains

  • Central SH2 domain binds pTyr on receptor
  • Flanking SH3 domains bind proline-rich PPII helices on the protein SOS
34
Q

SOS

A

is a guanine nucleotide exchange factor

  • Sos is a cytoplasmic protein and recruitment by Grb2 affects its localization to the membrane, which is the only place its target is found
  • The binding domain of Sos can fold up and autoinhibit the GEF domain when not bound to Grb2
  • Sos catalyzes the exchange of GTP for GDP in Ras
35
Q

Ras

A

is a key component of the MAP kinase cascade

All of the Ras Family Members Function as Binary Switches

36
Q

GTPase Activating Proteins (GAPs)

A

reduce Ras activation by
increasing its ability to hydrolyze GTP (~100-fold). Keep about 95% of Ras inactive in the absence of a signaling molecule.

Usually called Ras-GAPs.

37
Q

Guanine Exchange Factors (GEFs)

A

accelerate Ras activation by promoting the exchange of GTP for the bound GDP

ex. SOS

38
Q

The Importance of Being Turned Off (MAPK)

A
  • The intrinsic GTPase activity of Ras is weak and only hydrolyzes GTP slowly
  • GTPase Activating Proteins (GAPs, Ras-GAPs) act to enhance the weak GTPase activity of monomeric G proteins and promote conversion to the inactive state
  • All oncogenic forms of Ras analyzed from human cancers have mutations that interfere with the ability of Ras to interact with Ras-GAP
  • These oncogenic forms of Ras are mostly in the GTP-bound (active) state and continually signal to cells to promote cell division
39
Q

The MAP Kinase Cascade Linked to an RTK

A

Phosphorylation of Myc, Jun, & Fos leads to the transcription of genes involved in cell proliferation

40
Q

GTP-bound Ras interacts directly with the

A

ser/thr-specific protein kinase Raf (a MAP kinase kinase kinase) to activate it

41
Q

Raf

A

was first discovered as an oncogene;
oncogenic forms of Raf do not require signals from Ras, Ras-GEF or RTK to promote cell division.
(~75% of melanomas are caused by Raf)

42
Q

Raf only has one type of catalytic substrate -

A

the protein kinase Mek (a MAP kinase kinase)

43
Q

Mek

A

MAP kinase kinase

is unusual in that it contains both ser/thr protein kinase activity and tyrosine-protein kinase activity (on separate structural domains)

44
Q

Activated Mek

A

only has one catalytic substrate
family, MAP kinases, most commonly the protein kinase Erk

45
Q

Activation of MAP kinase requires

A

phosphorylation of a threonine and a tyrosine that are separated by only a single amino acid

MAP kinases have many target

46
Q

Activated MAP kinase can act on target proteins in both the cytoplasm and nucleus:

A

One action of MAP kinase is to activate gene transcription of genes involved in cell proliferation by phosphorylating and activating transcription activators

Biochem II Self Study (10 decks)

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