Transcript Slide 1

Ferchmin 2014
You will find some of the next slides apparently unrelated to the subject matter. Also there will be no continuity
between slides. Just focus on the concept presented in a given side and hopefully “by the end of the day” you
will see the application to glycogen regulation
Brief introduction to signal transduction needed to understand the regulation of glycogen
synthesis and breakdown. The regulation of the synthesis and breakdown of glycogen into
glucose is regulated in a complex manner with the intervention of hormones, receptors, Gproteins and protein kinases.
The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes.
There are many regulatory protein kinases in the cells, some of them phosphorylate other
protein kinases:
1) Cyclic AMP-dependent or PKA*
Since we had a reduction of lecture hours I will
2) Calcium/calmodulin-dependent (CaM II) or
PK II* that you will learn or already know about
assume
3) Phospholipid-dependent or PKC*
cell signaling. So, I will skip pages 1 to 6 which
4) Tyrosine kinases and receptor tyrosine kinases
or RTK
any way
were a preparatory discussion for the
5) Calcium dependent kinases
regulation of glycogen metabolism.
* These kinases phosphorylate serines or threonines.
There are Tyr and Ser-Thr protein kinases. The most remarkable example are the cascades of the
Mitogen Activated Kinases (MAP kinases or MAPK). You will see them in this and other courses.
This cascades are formed by MAPKKK MAPKK MAPK. In the case of ERK (Extracellular Regulated
Kinase) cascade. The first kinase (MAPKKK) is Raf. Raf is a serine/threonine kinase that is recruited
to the membrane by binding to active (thus, GTP-bound) G-protein Ras. The second is MEK
(MAPKK) is a dual specificity kinase since it can phosphorylate both threonine and tyrosine
residues, which it does on ERK (MAPK). ERK has cytosolic substrates but the most important role is
to be translocated to the nucleus where it phosphorylates specific substrates (proteins) leading to
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gene expression.
Simplified view of the MAP kinases
Go to page 6.
If you wish, you
could review on
your own the
skipped pages.
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PKA is the most important protein kinase in glycogen metabolism but a Ca2+-calmodulin
kinase also participates. In addition several kinases, regulated by insulin, like PKB/Akt and
glycogen synthase kinase-3 or GSK-3 are involved.
Where there are protein kinases there must be protein phosphatases.
1) Protein phosphatase I
2) Protein phosphatase II
There is also a protein phosphatase inhibitor that is activated by phosphorylation
by PKA on a threonine and prolongs the effect of PKA.
The interaction between kinases and phosphatases is regulated by targeting subunits. Docking proteins are modulatory
proteins that attach enzymes to specific targets, or specific locations. Often attaching to the membrane does the trick. These
targeting proteins ensure the fidelity of protein phosphorylation and facilitate that the proper protein kinase or phosphatase
quickly finds its substrate.
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CYCLIC AMP REGULATES THE SYNTHESIS AND BREAKDOWN OF
GLYCOGEN
β-Adrenergic receptor binds epinephrine in muscle and liver and a functionally similar
receptor binds glucagon in liver. These receptors are coupled to G-proteins. There are other
G proteins that are not receptor coupled. This binding activates the regulatory enzyme
adenyl cyclase. Adenyl cyclase synthesizes cyclic-AMP which is one of the so called second
messengers.
The message of cAMP is terminated by phosphodiesterase.
Calmodulin is a Ca2+ binding protein. It binds sequentially 4 Ca2+ per molecule and acts as
second messenger of Ca2+ . Often Ca2+ and cAMP regulate the same process.
There is a guanyl cyclase which synthesizes cGMP. The physiological role of cGMP is less
prominent than that of cAMP for glycogen metabolism.
The synthesis of cAMP is unfavorable, ΔGE◦'=1.6 Kcal/mole, however it is coupled to
the hydrolysis of PPi which releases about -4.6 Kcal/mole. Therefore, the net
synthesis of cAMP releases a total of -3 Kcal/mole, (+1.6 -4.6=3).
Interestingly, the hydrolysis of cAMP releases -12 Kcal/mole because it relieves the
strain imposed by the cyclic ester.
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G proteins are a family of GTP binding modulators
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Glucagon receptor
and beta
adrenergic
receptors are
functionally almost
equal. Glucagon
receptors are
present in liver and
adrenergic in liver
and the periphery.
Representative pathway for the activation of cAMP-dependent protein kinase, PKA. In this example glucagon binds to
its' cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a
receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the αsubunit dissociates and binds to and activates adenylate cyclase. Adenylate cyclase then converts ATP to cyclic-AMP
(cAMP). The cAMP binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic
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subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the
catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.
After this hasty and nasty introduction to cell signaling we will study the regulation
of glycogen synthase and glycogen phosphorylase and a regulatory enzyme called
phosphorylase b kinase. For the three enzymes we will use the diagram shown below.
Please, understand the diagram. Later we will integrate all this in a graph.
The granule of glycogen contains the three enzymes mentioned above plus other
regulatory enzymes.
This cartoon shows the
phosphorylation and
dephosphorylation of
glycogen synthase
Let me introduce the
idea of the “monster”
that activates adenylate
cyclase (adenylyl cyclase)
and antagonizes
fructose-2,6-di-phosphate.
But active in the presence of
glucose-6-phosphate
Glycogen synthase can be D, or dependent on glucose-6-P, or I, independent of the presence
of glucose -6-P. Please, remember that the immediate precursor of glycogen is UDP-glucose
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not glucose-6-P. The latter is only an allosteric regulator of glycogen synthase.
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This enzyme is
regulatory.
Does not affect
“real”
metabolites.
On top of it its
is “misnamed”.
See below.
Glycogen phosphorylase b kinase also phosphorylates the synthase and should actually be
called synthase phosphorylase b kinase. The phosphorylation of the alpha subunit regulates
the dephosphorylation of the beta subunit. The delta subunit is calmodulin. The direct
interaction of Ca2+ with calmodulin activates this enzyme. This effect is specially relevant in
muscle.
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Integration of the regulation of glycogen synthesis and breakdown
Adenylyl Cyclase
Glucagon
Epinephrine
Receptors
cAMP
ATP
AMP
R2C2
R2(cAMP)4
Phosphodiesterase
2C
ACTIVE PKA
Glycogen synthase
Slides 7 to 9 are conceptually merged in slide 10.
Slides 10, 11 and 12 introduce in a stepwise
manner the regulation of glycogen metabolism
by epinephrine (in muscles and liver) and by
glucagon in liver. Slides 13 and 14 introduce
the role of insulin. All of that is integrated in
slide 15. If you study sequentially all the
slides (10 to 15) you will be able (hopefully) to
understand the regulation of glycogen metabolism .
Phosphorylase b
Kinase
Glycogen Phosphorylase
activation
inhibition
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Integration of the regulation of glycogen synthesis and breakdown
Adenylyl Cyclase
Glucagon
Epinephrine
Receptors
cAMP
ATP
AMP
R2C2
R2(cAMP)4
Phosphodiesterase
2C
ACTIVE PKA
Phosphorylase b
Kinase
Glycogen synthase
Glycogen Phosphorylase
Protein Phosphatase
activation
Protein Phosphatase Inhibitor
inhibition
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Integration of the regulation of glycogen synthesis and breakdown
Adenylyl Cyclase
Glucagon
Epinephrine
Receptors
cAMP
ATP
AMP
R2C2
R2(cAMP)4
Phosphodiesterase
Ca2
2C
ACTIVE PKA
+
Phosphorylase b
Kinase
Glycogen synthase
Glycogen Phosphorylase
Protein Phosphatase
activation
Protein Phosphatase Inhibitor
inhibition
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Insulin activates glycogen synthesis
You must know the
mechanism of insulin
receptor. It is by Tyr
phophorylation on the
receptor and on its
substrates
13
Insulin stimulates glucose transport in muscle and adipose cells by stimulating
translocation of glucose transporter 4 (GLUT4) to the plasma membrane.
14
Integration of the regulation of glycogen synthesis and breakdown
Insulin-R
PI3-K
Adenylyl
Cyclase
Remember
that
ATP
(-1) x (-1)=+1
or inhibition of
cAMP is as
inhibition
good as activation
PKB/Akt
R2C2
R2(cAMP)4
Glucagon
Epinephrine
Receptors
Heart,
muscle and
other
AMP
Phosphodiesterase
GSK-3
Ca2
2C
ACTIVE PKA
+
Phosphorylase b
Kinase
Glycogen synthase
Glycogen Phosphorylase
Protein Phosphatase
activation
Protein Phosphatase Inhibitor
inhibition
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Akt inhibits glycogen synthase kinase 3 (GSK-3) which then stops inhibiting the glycogen synthase
We will leave glycogen metabolism and return to glycolysis,
fructose-2,6-bisphosphate, xylulose-5-P, and expand the regulatory
big picture
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Take home message.
When you are chased by
a monster your liver
won’t synthesize
glycogen.
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2) Nonoxidative
steps of
pentose phosphate shunt
Misplaced
slide?
No, it is integration
of glucose metabolism
with lipid synthesis
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Regulation of fructose-2,6-bis phosphate synthesis and break down
HEXOSES
Fructose-6-P
Fructose-2,6-bis-phosphate
ATP
ADP
ATP
Phosphofructokinase-2
Pi
PP2A
Bifunctional enzyme.
Phosphorylated is phosphatase
Dephosphorylated is kinase
Lipogenesis
PKA
PKA is just
that, protein
kinase A
Fructose-2,6-bis-phosphatase
Do you
remember
xylulose-5phosphate?
PP2A is
protein
phosphatase
2A
Fructose-6-P
Pi
H2O
Fructose-2,6-P
cAMP
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This “repeated” slide was placed for you to practice the metabolism of fructose-2,6-P
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Role of 2,6-Fructose bisphosphate and phosphofructokinases-1
and -2
Phosphofructokinase-1 is the well-known glycolytic enzyme; phosphofructokinase-2 is
exclusively a regulatory enzyme. PFK-2 has kinase activity when nonphosphorylated and
catalyzes the phosphorylation of fructose-6-P in carbon 2.
When phosphorylated phosphofructokinase-2 has phosphatase activity and catalyzes the
dephosphorylation of carbon 2.
The role of 2,6-FDP is to "convey" to the liver cells that there is plenty of hexoses and that
glycolysis has to be activated to support fatty acid synthesis. At the same time,
gluconeogenesis has to be inhibited. In liver glycolysis is inhibited by cAMP, which
indirectly inhibits phosphofructokinase-1 (by lowering 2,6-FDP and activating 1,6-FDP
phosphatase) and liver pyruvate kinase (as seen above). In muscle, cAMP activates
glycolysis by activation of glycogenolysis. This difference in regulation reflects the different
roles of glycolysis in both organs.
You should recall from the regulation of glycolysis that 2,6-FDP activates the hepatic PFK1 by removing the inhibitory effect of ATP. Also 2,6-FDP enhances the action of PFK-1 by
inhibition the 1,6- FDP phosphatase.
In cardiac muscle it seems that phosphofructokinase-2 is a different isozyme than
in liver and the phosphorylated muscle phosphofructokinase-2 is active thus
allowing cyclic-AMP to activate glycolysis.
21
Pancreatic β-cells and hepatocytes
have the same glucokinase
and GluT2
transporter.
Why?
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Abbreviated glycolysis, gluconeogenesis, and pentose shunt pathways and roles of Xu5P
and Fru-2,6-P2 in activation of PP2A and PFK, respectively.
Kabashima T et al. PNAS 2003;100:5107-5112
©2003 by The National Academy of Sciences
Veech RL (2003) A humble
hexose monophosphate
pathway metabolite regulates
short- and long-term control of
lipogenesis. Proc Natl Acad Sci
U S A 100:5578-5580.
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