Transcript Poster for RCPSC mee.. - University of Alberta

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
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 (6). 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 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 non-conserved 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.
We have identified glucosylceramide as a novel inhibitor of PP1c and PP2Ac.
Mutagenesis studies of PP1c have shown that residues in both the β12-β13 loop and the
hydrophobic groove are important for the inhibition of PP1c by glucosylceramide.
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
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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.
120
90
PP2A
60
PP1
30
0
0
10
20
Future Directions
30
[Glucosylceramide] (uM)
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).
Figure 4 (left): Differences in
sequence in the β12-β13 loop region
of PP1, PP2A and PP2B
(Calcineurin). The β12-β13 loop
corresponds to residues 273-277 in
PP1c.
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).
Effect of Glucosylceramide on PP1c Activity
150
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: 4407844082 (November 2001).
120
wild type
loop mutant
Y134A
90
60
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
(February 2005).
6.
Yusuf A. Hannun and Chiara Luberto. Ceramide in the Eukaryotic Stress Response. Trends Cell Biol. 10: 73-80 (February 2000).
30
0
0
10
20
30
[Glucosylceramide] (uM)
Figure 1: The PP1 inhibitor
clavosine (left), and the
sphingolipid glucosylceramide
(right).
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
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.
Figure 5: The β12-β13 loop as well as residue Tyr-134 of the PP1c hydrophobic groove, are important in
binding of GlcCer to PP1c.
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: GlcCer in rat sympathetic neurons
causes a decrease in PP1 activity in cell lysates
from rat sympathetic neurons.
PP1 Activity (x106 U/g protein)
Figure 2: Crystal structure of PP1c
bound to clavosine (Maynes, JT et al,
unpublished results. The residues of the
β12-β13 loop as well as the residue
Y134 of the hydrophobic groove of the
enzyme are indicated in green.
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. (** refers to p<0.005).
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 β12-β13 loop (left)
and Y134 residue (bottom middle) of PP1c (Maynes, JT et al, unpublished data).