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A Novel Lipid Inhibitor of Protein Phosphatase-1
Kathleen R. Perreault*, Brian Dembinski^, Jason T. Maynes*, Michael N. G. James, Elena Posse de Chaves^, and Charles F. B. Holmes*
From the Canadian Institutes of Health Research, Group in Protein Structure and Function and the Signal Transduction Research Group, the Departments of Biochemistry* and Pharmacology^,
Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Introduction
Conclusions
Results
Ceramide is a sphingolipid second messenger produced in response to cellular stress via activation of
sphingomyelinases. Agonists that cause cellular production of ceramide include cytokines (TNF, Fas), agents of
environmental stress (heat, UV irradiation), and chemotherapeutic agents. The accumulation of ceramide activates
JNK/SAPK, PKCζ, caspases as well as PP1 and PP2A. Substrates of PP1 and PP2A that are dephosphorylated in
response to either ceramide-inducing agonists or addition of exogenous ceramide include c-jun, SR proteins,
retinoblastoma protein, PKB/Akt1, protein kinase Cα and Bcl-2.
Glucosylceramide (GlcCer) is a metabolite of
ceramide produced by the glycosylation of the 1-hydroxyl group of ceramide by the enzyme Glucosylceramide
Synthase (GCS) (Figure 1). Given the similarities in structure between the natural product inhibitors of PP1, the
clavosines and calyculins, and the sphingolipid GlcCer, we hypothesized that GlcCer may affect PP1c activity by
binding to the catalytic subunit in a similar fashion. Using radiolabelled glycogen phosphorylase a, a physiological
substrate of PP1, we found that GlcCer inhibited PP1 activity in vitro. Using site-directed mutagenesis of the PP1
catalytic subunit (PP1c), we determined that the β12-β13 loop (Figures 2 and 4), an unstructured chain of five nonconserved amino acid residues present in PP1, PP2A and PP2B, is important for binding of GlcCer to PP1c. Ceramide
activation of PP1c is unaffected by mutations in this region. We also found that mutation of Tyr-134, an amino acid
residue present at the interface between the hydrophobic groove and the active site of PP1c, greatly decreases the
potency of GlcCer inhibition. Finally, we used lysates of live cells with accumulated GlcCer to show that endogenous
PP1 activity is also decreased in the presence of GlcCer.
Figure 3: GlcCer inhibits both PP1c and PP2Ac.
Inhibition of PP1c (IC50~5 μM) is approximately 3
times more potent than inhibition of PP2Ac
(IC50~15 μM).
Effect of Glucosylceramide on PP1c and
PP2Ac Activity
Percent of Control
Release
Protein phosphatase-1 (PP1) is a Ser/Thr phosphatase of the PPP family, which is also comprised of PP2A,
PP2B (Calcineurin), PP4, PP5, PP6 and PP7. PP1 activity is regulated by many endogenous protein inhibiting/targeting
subunits. A number of structurally diverse natural product toxins are also potent inhibitors of PP1 activity (Figure 1).
Despite the structural diversity of these toxins, they have several coarsely similar features that aid in binding to PP1:
hydrogen bonding to specific conserved residues in close proximity to the site of enzymatic activity, acidic groups that
interact with conserved basic amino acids within the active site, and hydrophobic regions that allow binding at the
hydrophobic groove adjacent to the active site (1-3).
Results
150
120
90
PP2A
60
PP1
0
10
20
[Glucosylceramide] (uM)
Figure 4 (above): Differences in sequence in the ß12-ß13 loop region of PP1, PP2A and PP2B (Calcineurin). The ß12-ß13
loop corresponds to residues 273-277 in PP1c. Because this is a non-conserved sequence between PP1 and PP2A, located
close to the active site and shown to be important in interaction with other PP1 inhibitors, we examined it’s importance in
GlcCer inhibition of PP1 and PP2A. We mutated residues 273-277 in PP1c (CGEFD) to the corresponding residues in
PP2B, and examined inhibition of the mutant enzyme by GlcCer (below). In addition, we used a mutation of PP1c in the
hydrophobic groove to determine the importance of this region in binding to GlcCer.
We hope to carry out studies on the effect of endogenous and exogenous
glucosylceramide on the phosphorylation states of PP1 and PP2A substrates. Because
glucosylceramide has been shown to accumulate in multidrug-resistant cancer cell lines like
the KB cell lines used in our study, we are particularly interested in the phosphorylation
state of proteins involved in cell cycle arrest and apoptosis. Previous studies have shown
that treatment of sympathetic neurons with ceramide (PP1 activator) blocks
hyperphosphorylation of pRB (retinoblastoma gene product), and therefore we hypothesize
that we may see hyperphosphorylation of pRB upon treatment of neurons with GlcCer (5).
References
Effect of Glucosylceramide on PP1c Activity
Figure 5: The ß12-ß13 loop as well as
residue Tyr-134 of the PP1c hydrophobic
groove, are important in binding of GlcCer to
PP1c. Mutating the residues of the ß12-ß13
loop of PP1c to the corresponding residues in
PP2B caused a 3-fold decrease in inhibition
by GlcCer, resulting in an IC50 of ~15 μM.
Mutating the hydrophobic groove residue Tyr134 to Ala caused an even more potent
decrease in inhibition (IC50>20 μM).
150
120
wild type
loop mutant
Y134A
90
60
30
0
0
10
20
30
Figure 6: Glucosylceramide has been shown to
accumulate in several multidrug-resistant (MDR)
cancer cell lines (4). We examined PP1 activity and
GlcCer content in multidrug-resistant human
epidermoid carcinoma cells. We found these cells
have increased glucosylceramide content and
decreased PP1 activity. For Figures 6 and 7,
statistically significant results compared to untreated
cultures are indicated ** (p<0.005).
1. Kathleen R. Perreault, Jason T. Maynes, Maia M. Cherney, Hue Anh Luu, Michael N. G. James, and Charles F. B.
Holmes. Crystal Structure and Mutagenesis of a Protein Phosphatase-1:Calcineurin Hybrid Elucidate the Role of
the ß12-ß13 Loop in Inhibitor Binding. J. Biol. Chem. 279: 43198-43206 (October 2004).
2. Jason T. Maynes, Katherine S. Bateman, Maia M. Cherney, Amit K. Das, Hue Anh Luu, Charles F. B. Holmes, and
Michael N. G. James. Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1. J.
Biol. Chem. 276: 44078-44082 (November 2001).
3. Charles F. B. Holmes, Jason T. Maynes, Kathleen R. Perreault, and Michael N. G. James. Molecular Enzymology
Underlying Regulation of Protein Phosphatase-1 by Natural Toxins. Curr. Med. Chem. 9: 1981-1989 (November
2002).
4. Yaakov Lavie, Hui-ting Cao, Stuart L. Bursten, Armando E. Giuliano, and Myles C. Cabot. Accumulation of
glucosylceramides in multidrug-resistant cancer cells. J. Biol. Chem. 271:19530-6 (August 1996).
5. Greg Plummer, Kathleen R. Perreault, Charles F. B. Holmes, and Elena I. Posse de Chaves. Activation of
Serine/Threonine Protein Phosphatase-1 is Required for Ceramide-Induced Survival of Sympathetic Neurons.
Biochem. J. 385: 685-693 (September 2004).
Cell Type
KB-3-1
PP1 Activity
4.6±0.3
(x107 U/g protein)
KB-V.01
KB-V.1
4.0±0.2*
3.6±0.1**
KB-V1
3.4±0.1**
GluCer content
(TLC)
**
Figure 7: Accumulation of GlcCer in live cells
causes a decrease in PP1 activity.
Rat
sympathetic neurons were incubated overnight
with the indicated concentrations of GlcCer and
Cer. The cells were lysed, and PP1c activity in
the lysates was measured using phosphorylase a
as a substrate. We found that PP1 activity
decreased with increasing exposure to GlcCer.
PP1 Activity (x106 U/g protein)
Figure 2: Crystal structure of
PP1c bound to clavosine. The
residues of the ß12-ß13 loop as
well as the residue Y134 of the
hydrophobic groove of the
enzyme are indicated in green.
The methylated rhamnose
moiety is in close proximity to
the ß12-ß13 loop.
The
hydrophobic groove, which
contains the residue Y134
binds the polyketide chain of
the inhibitor. We propose that
these regions are also important
in forming interactions with the
C18-sphingosine moiety of
ceramide and GlcCer.
Studies using lysates from live cells show that this inhibition is not purely an in vitro
phenomenon, as endogenous PP1 activity is also affected by an increase in
glucosylceramide.
Future Directions
30
[Glucosylceramide] (uM)
Figure 1: The PP1 inhibitors clavosine (left), and the sphingolipid glucosylceramide (right).
We have identified glucosylceramide as a novel inhibitor of PP1c and PP2Ac.
Mutagenesis studies of PP1c have shown that residues in both the ß12-ß13 loop and the
hydrophobic groove are important for the inhibition of PP1c by glucosylceramide.
30
0
Percent of Control Release
Reversible protein phosphorylation is an integral mechanism of signal transduction in many important cellular
processes, including, but certainly not limited to, mitogenesis, apoptosis, and regulation of gene expression. In the
human genome, the ratio of Ser/Thr protein kinases to Ser/Thr protein phosphatases is approximately 8:1. The
corollary of this unbalanced ratio is that an individual phosphatase is responsible for dephosphorylating a broad range
of substrates, and therefore must be promiscuous with respect to substrate specificity. To compensate for this relative
lack of specificity, Ser/Thr phosphatases are regulated by a large number of inhibitory and targeting subunits, which
serve to direct their activity towards the appropriate substrate.
3
2
**
**
20
30
1
**
0
control
5
10
C8-GlcCer
(M)
40
C6- Cer
Figure 8: A close-up view of the proximity of bound clavosine to the ß12-ß13 loop (left)
and Y134 residue (bottom middle) of PP1c. The blue circles are the catalytic manganase
ions in the active site of the enzyme.