Download Activation of protein kinase A and protein kinase C via intracellular

Biochemistry 302 – Examination #4 – May 10, 2002
Student’s Full Name ________________________________________________
This examination is worth 75 points. You should be able to finish the exam in 1.5 hours and answer each question
completely. Please be succinct and clear in your answers. More space is provided than will be necessary to answer
the questions. Partial credit will be given where appropriate.
GOOD LUCK!!!
( 8 pts) 1.
You are studying an intracellular signalling cascade in which the activation of protein kinase A (PKA)
appears to have no effect on gene transcription, whereas its effects on other intracellular events appear
normal.
a.
How would you begin to describe this abnormality? (3 points)
PKA’s effects of gene transcription require phosphorylation of CREB (cAMP response element
binding protein), such that it can bind to the cAMP response element, expressed by some genes,
and recruit other transcriptional activators to initiate transcription. So…you should begin to
look for potential problems in that pathway (e.g. for example, perhaps the CREB cannot be
phosphorylated).
b.
Activation of protein kinase C (PKC) via intracellular signalling events can also result in
increased protein synthesis. Describe how PKC can alter gene transcription rates. (5 points)
1. PKC directly activates MAP kinase (ERK) which can then phosphorylate and thereby activate
appropriate transcription factors, which it recognizes as substrates.
2. PKC phosphorylates IB relieving its inhibition of the transcription factor NF-B.
(15 pts) 2.
The binding of the cytokine, TNF, or the growth factor derived from platelets, platelet-derived growth
factor (PDGF), to their respective, individual receptors leads to activation of phospholipase C
(PLC).
a.
Describe the structural features of PLC and the activated receptors that are required for PLC
activation. (4 points)
PLC contains SH2 and SH3 domains which are required for its binding to distinct
phosphotyrosines (docking sites) on the activated receptor.
b.
Discuss the two mechanisms unique to activation of the different TNF and PDGF receptors
that occur subsequent to ligand binding, which result in PLC activation. (6 points)
Receptor protein tyrosine kinases vs. Receptor associated tyrosine kinases
PDGF binding to its receptor leads to receptor activation and dimerization with the subsequent
expression of the receptor’s tyrosine kinase activity. Each subunit of the dimerized receptor
cross-phosphorylates its partner with autophosphorylation occurring subsequently. Select
phosphotyrosines providing appropriate docking sites for a variety of SH2 and SH3 domain
containing proteins.
TNF binding to its receptor leading to receptor dimerization and recruitment of the appropriate
cytoplasmic Tyr-kinase which phosphorylates the receptor’s cytoplasmic tails on again select
phosphotyrosines, providing appropriate docking sites for a variety of SH2 and SH3 domain
containing proteins.
c.
Protein dimerization is a common theme in receptor-mediated intracellular events. What
dimerization events are required for the activation of gene transcription that occurs subsequent to TNF
binding to its receptor? (5 points)
As stated above, receptor dimerization results in its appropriate phosphorylation by an
associated cytoplasmic Tyr-kinase. Appropriate phosphotyrosines recuit STAT via its SH2 and
SH3 binding domains. STAT will be phosphorylated facilitating its release from the receptor
and its ability to dimerize with another activated STAT. The STAT dimer moves to the nucleus
to bind its target gene.
(10 pts) 3.
Discuss, in order of their occurrence, all the allosteric events required for activation of PKA by
epinephrine binding to its receptor on adipocytes.
Epinephrine binding to the -adrenergic receptor on adipocytes  a conformational change in
the receptor at its allosteric site  conformational change in the bound, inactive heterotrimeric
G protein   in the ability of the G protein  subunit to exchange its bound GDP for GTP 
conformational change in the G protein and  subunit dissociation from the  subunits.
Interaction of the activated  subunit with adenylate cyclase  conformation change in the
enzyme that stimulates its activity to convert ATP to cAMP. The formed cAMP allosterically
regulates PKA by binding to the appropriate sites on the regulatory subunits  allosteric
changes in the enzyme that allow dissociation of the catalytic subunits from the regulatory
subunits.
Long winded way of saying: There are allosteric changes in 1) the receptor; 2) the bound G
protein; 3) the “activated subunit; 4) adenylate cyclase and, 5) PKA via formed cAMP.
(12 pts) 4.
The direct activation of a protein kinase C isoform in platelets by phorbol myristate acetate (PMA)
results in the specific phosphorylation of a single 80 kDa protein. Interestingly, thrombin activation of
platelets via PAR1 results in the phosphorylation of the same 80 kDa protein and the phosphorylation
of select Ser/Thr residues on the intracellular protein, plekstrin.
a.
Based on this information, describe the signalling pathway initiated by thrombin, its major
constituents and the sequence of events leading to phosphorylation of both plekstrin and the 80 kDa
protein. (8 points)
Thrombin interaction with its receptor leads to activation of the G protein, Gq, which activates
phospholipase C. Functional phospholipase C catalyzes the hydrolysis of phosphoinositol-4,5bisphosphate (PIP2), residing in the inner membrane, to form the second messengers inositol1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to its receptor on the endoplasmic
reticulum to effect Ca2+ release which in concert with DAG will activate protein kinase C (PKC).
The increased intracellular Ca2+ will bind to CaM as detailed above to effect phosphorylation of
plekstrin, whereas PKC will catalyze the phosphorylation of the 80 kDa protein, either directly
or perhaps indirectly through activation of another kinase.
b. (4 pts) How is this particular signalling pathway and its downstream effects “down-regulated?”
1. Ca2+ is rapidly pumped out of the cytoplasm via plasma membrane- , ER- and mitochondrialassociated pumps.
2. IP3 is rapidly dephosphorylated by appropriate phosphatases.
3. Diacylglycerol (DAG) is rapidly hydrolyzed
4. Substrates phosphorylated by PKC or CaM kinases are dephosphorylated by Ser/Thr
phosphatases.
5. The receptor will most likely be desensitized by GRK-catalyzed phosphorylation  arrestin
binding and perhaps, subsequent receptor-mediated endocytosis.
6. The GTPase activity of the  subunit will hydrolyze the bound GTP to GDP thereby downregulating its activity.
( 7 pts) 5.
As you are aware, estrogen interacts with an intracellular receptor, in contrast to a transmembrane
receptor.
a. (1 pt) Since estrogen reaches all tissues via passive transport from the blood stream, how is it that
only a very few tissues, such as vascular endothelial cells and cells of the uterus, respond to estrogen?
The responsive cells express the estrogen receptor. Non-responsive cells, which do not contain
the receptor, metabolize the cytoplasmic estrogen.
b. (4 pts) What are the structural and functional domains of the estrogen receptor?
Steroid receptors are composed of three functional domain. The COOH-terminus contains the
unique hormone binding site as well as sites for receptor dimerization. The middle domain
contains the DNA binding site. The NH2-terminus contains regions essential for transcriptional
activation.
c. (2 pts) How does the occupied estrogen receptor function to induce a cellular response?
The occupied receptors dimerize becoming an “active” transcription factor capable of binding to
its hormone response element in the appropriate target gene to alter gene transcription rates.
(16 pts) 6.
During your predoctoral studies, you isolated and immortalized a vascular smooth muscle cell line that
displays uncontrolled cell growth when exposed to growth factors such as PDGF. One of several
possibly dysfunctional proteins could account for this abnormal cellular phenotype. In the list below,
underline the potential abnormal proteins and for those underlined, briefly state how their abnormal
behavior could contribute to uncontrolled cell growth.
You’re looking for those proteins which are part of a growth factor-initiated Receptor Protein
Tyrosine Kinase-mediated intracellular signaling pathway
IRS-1 (required for insulin-induced signaling)
Janus kinases (JAKs) (part of the Tyr kinase associated receptor signaling pathway)
Raf – MAP kinase kinase kinase; normally activated by ras, but in this case may be “always” on
Ras – monomeric GTPase that activates Raf; Subsequent to its activation its associated GTPase
may not function or, alternatively its associated GEF may always by bound keeping it in a
continually activated state.
Ras-GAP – The GTPase activating protein of ras; it may not be able to function to turn off ras.
ARK (GRK for the -adrenergic G protein coupled receptor)
Ser/Thr phosphatases – required to dephosphorylate kinases in the MAP kinase cascade; if not
functioning properly it will be difficult to down-regulate that cascade.
STATs (part of the Tyr kinase associated receptor signaling pathway)
MEK – MAP kinase kinase; if in a continuously activated state it would continue to
phosphorylate MAP kinase
Grb2 – Adaptor protein that binds to the phosphorylated PDGF receptor to provide the binding
site for ras-GEF; may lock ras-GEF into a continuously active state
MAP kinase – When phosphorylated by MAPKK, will subsequently phosphorylate appropriate
transcription factors leading to increased transcription rates and cell growth; when locked in an
activate conformation, for whatever the reason, uncontrolled cell growth will result
Sos – ras GEF (guanine nucleotide exchange factor); facilitates the exchange of GTP for bound
GDP on ras; may lock ras into a perennially activated state
PLC (activated by Gq)
cAMP phosphodiesterase (hydrolyzes cAMP formed in response to adenylate cyclase activation
mediated by the  subunit of Gs.
( 7 pts) 7.
You have done an experiment to assess the binding parameters governing the interaction of 125Ilabeled PDGF (125I-PDGF) with its receptor on vascular endothelial cells under conditions in which
you can accurately quantify the amount of ligand bound with increasing concentrations of added
ligand. Your basic experimental protocol is to mix various concentrations of 125I-PDGF with vascular
endothelial cells (1 x 108 cells/mL) for 10 min at 37C, pellet the cells by centrifugation through oil
and measure the radioactivity in the pellet to quantify bound ligand. The data you obtained are shown
graphically below as a Scatchard plot, which is described by the following equation:
b/f = -1/Kd•b[ligand] + [R]T/Kd
WE WILL ANSWER THIS QUESTION IN THE REVIEW
SESSIONS SCHEDULED FOR MON (4/5) and TUES (4/6)
Based on the data shown, calculate the total receptor concentration, [R]T, present in the assay.
Calculate the dissociation constant (Kd) defining PDGF binding to its receptor on vascular endothelial cells?
Assuming a 1:1 binding interaction between PDGF and its receptor, calculate the number of receptors per cell.
BONUS – 5 points
You have just demonstrated that the serine protease plasmin will activate human platelets through a
mechanism that requires the proteolytic activity of the enzyme. You hypothesize that plasmin is activating
platelets either through cleavage of PAR1, or PAR3, or both PAR1 and PAR3, since these two receptors are
expressed on human platelets. What experiment or experiments could you design that would allow you to
distinguish between these various possibilities.