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