Phosphorylase Kinase






Converts phosphorylase b  phosphorylase a
High abundance protein (0.5% in muscle)
Native MWt 1.3 x 106 Da
4 different subunits: probably 444 4
-subunit (45 kDa) has the full protein kinase activity
subunits (140 and 120 kDa, respectively) are inhibitory
subunits unless phosphorylated by Protein Kinase A
 -subunit is the catalytic subunit which catalyses the
phosphorylation of phosphorylase b at Ser14
 -subunit is calmodulin which confers Ca2+ sensitivity to the
phosphorylase kinase – the enzyme becomes activated when
[Ca2+]cyt increases e.g. in muscle contraction

Calmodulin
 monitors the Ca2+ levels in the cytoplasm
 can exist as a free form or as a subunit of a heteromeric protein
 is a highly conserved protein which acts as a Ca2+ activated switch
in many organisms regulating many different cell functions
 it has 4 Ca2+ binding sites per mol with a Kd (dissociation
constant) of ~ 1 µM – consistent with the levels of Ca2+ in
contracting muscle
 participates in numerous regulatory processes, including muscle
contraction and phosphorylase kinase activation
 the binding of Ca2+ induces a conformational change in
calmodulin, exposing a Methionine-rich, hydrophobic domain
which in turn, can then bind to and alter the catalytic function of
the -subunit and other regulatory proteins.
Physiological significance in muscle
 muscle contraction is stimulated by a transient increase in [Ca2+]cyt
from 0.1 µM to 1 – 10 µM
 the rate of glycogen breakdown (and thus the provision of ATP by
anaerobic glycolysis) is linked to muscle contraction
 for phosphorylase kinase to be fully activated the subunits
must also be phosphorylated by PKA, which, in turn, is activated
by adrenaline via cAMP in muscle
D Davies BS2510 2003
Protein Phosphatase 1 (PPase 1)
 reverses the effect of protein kinases on glycogen metabolism
 the steady-state activity of many regulatory enzymes depend on
the fraction of the enzyme in the phosphorylated and
dephosphorylated states
 this is in turn dependent on the relative activities of protein
kinases and protein phosphatase
 about 1/3 of all mammalian proteins have covalently-bound
phosphates which may impact on some aspect of their regulation
 there are about 1,000 different protein kinases encoded in the
human genome and about 500 different protein phosphatases
 Protein Phosphatase nomenclature is rather arbitrary, based
mainly on the order in which the have been discovered: the most
important form as far as glycogen metabolism is concerned is
PPase 1, with a lesser role played by PPase 2A
 These enzymes hydrolyse specific phosphate residues covalently
bound to Serine or Threonine, thus reversing the effects of the
protein kinases
 PPase 1 brings about the dephosphorylation and inactivation of
Phosphorylase a and of phosphorylase kinase (subunits)
and the dephosphorylation and activation of Glycogen Synthase.
Regulation of PPase 1
 In turn the activity of PPase 1 is regulated by two different
proteins
 PPase Inhibitor 1 (Inhibitor 1) and G-subunit
Inhibitor 1
 Inhibitor 1 (found in both liver and muscle) is yet another
example of a protein regulated by PKA and PPase 1 although in
this case the protein is phosphorylated on a Threonine residue

 The protein is only functional (i.e. inhibitory) when it is
phosphorylated.
 It inhibits PPase 1

 The concentration of cAMP in the cell controls the fractions of
Phosphorylase and Glycogen Synthase which are in the active
form by activating PKA and by inhibiting PPase 1. The two effects
reinforce one another.
G-subunit
 G-subunit (Glycogen-targeting Subunit) occurs in different forms
in muscle (160 kDa) and liver (33 kDa) ( nothing to do with heterotrimeric,
plasma membrane associated G-proteins involved in signalling!)
 G-subunits target PPase 1 to glycogen and therefore to the
enzymes of glycogen metabolism
 One form of PPase 1G is far more active than other forms, this
consist of the catalytic subunit and the G-subunit

 Protein kinase A can also phosphorylate G-subunit leading to the
release of PPase 1 into the cytosol and the inactivation of the
enzyme is completed by Inhibitor 1. The reason for this
mechanism is probably to prevent the uncontrolled
dephosphorylation of cytosolic proteins by PPase 1 – the enzyme is
only active when associated with the Glycogen

 The dissociation of the G-subunit/PPase 1 complex is dependent
on the phosphorylation of G-subunit at site 1 and this is reversed
by PPase 2A. This occurs quickly once the adrenalin has exerted
its desired effect, G-subunit can recombine with the catalytic
subunit, PPase 1 is activated and GS is activated, phosphorylase
inactivated and glycogen synthesis can recommence

 In times of plenty, insulin stimulates Glycogen Synthase via the
activation of an insulin-stimulated protein kinase which leads to
G-subunit being phosphorylated at site 2, which results in the
activation of PPase 1 and Glycogen synthesis occurs.
Regulation of Glycogen Metabolism in the liver

There are many points of similarity with muscle where these have
been studied and some differences reflecting the different function
of glycogen metabolism in the two tissues(See below)
 G-subunit is much smaller (33kDa, 284 aa), binds PPase 1 at a
sequence RVSF near the N-terminal, binds glycogen at residues
144-231, and phosphorylase a near the C-terminal. But there is no
association with the ER and G-subunit is not regulated by
phosphorylation/dephosphorylation in liver.




PP1
Glycogen
56 –64
144-231
Phosphorylase a
269-284
Glucagon mediates increased cAMP in liver, adrenalin and
noradrenalin work via changes in IP3 and Ca2+.
Liver glycogen is used to buffer blood glucose. The liver has a
specific Glucose-6-phosphatase and Glucokinase to regulate
uptake and export of the sugar.
When glucose is infused into the liver, glycogen synthesis only
starts after glycogen degradation has stopped. Similarly when
cAMP is infused Glycogen Synthase is only switched off when
phosphorylase has been fully activated.
In the liver PPase 1 is strongly bound to phosphorylase a. When
glucose level rises, glucose binds to phosphorylase a ,this changes
its conformation making it a better substrate for PPase 1. This
results in the inactivation of phosphorylase and the release of
PPase 1 which can then dephosphorylate and activate glycogen
synthase