This mini-review highlights the involvement of PP2C (protein phosphatase type 2C) family members α and β in apoptosis. The activity of these isoenzymes can be stimulated by unsaturated fatty acids with special structural features, e.g. oleic acid. Those fatty acids capable of activating PP2Cα and PP2Cβ in vitro induce apoptosis in various cell types as shown here for neurons and endothelial cells. Using RNA interference to reduce the amount of PP2Cα and PP2Cβ results in cells significantly less susceptible to the apoptotic effect of oleic acid. Increased endothelial cell death is considered to be an initial step of atherogenesis. Thus activation of PP2C by physiological unbound (‘free’) unsaturated fatty acids (liberated from lipoproteins) could represent a crucial mechanism in the development of atherosclerosis.

Introduction

Reversible and mostly multiple phosphorylation of proteins is a crucial mechanism that is used in eukaryotic organisms to regulate all kinds of cellular processes. Kinases and phosphatases manipulate the phosphorylation status of their substrates, thereby regulating regulatory proteins. Quite often, kinases and phosphatases themselves are additionally subject to reversible phosphorylation. Approximately 500 kinases and 150 phosphatases are encoded by the human genome (for reviews on reversible phosphorylation, see [13]).

PTPs (protein tyrosine phosphatases) are one huge branch of protein phosphatases. The other superfamily acts on serine/threonine residues and can be subdivided into two categories. (i) PPP (phospho protein phosphatase) members include types 1, 2A, 2B, PP4, PP5 and PP6 and many more, which are defined by distinct amino acid sequences. (ii) PPM (protein phosphatase magnesium-dependent) enzymes are characterized by their requirement for Mg2+ or Mn2+ ions for activity. PPM comprises PP2C (protein phosphatase type 2C) and pyruvate dehydrogenase phosphatase (for a classification of protein phosphatases, see [46]). Until now, 14 different PP2C genes have been identified in mammalian cells with distinct localization, molecular mass and function, yet all are characterized by the requirement of Mg2+ or Mn2+ ions for activity. Sequence analysis and alignments revealed the conservation of 11 motifs within eukaryotic PP2C family members [7]. The purpose of this brief review is to highlight some recent advances in our understanding of the physiological and pathophysiological functions of PP2Cα and PP2Cβ.

PP2Cα and PP2Cβ

Identification of PP2Cα (PPM1A) and PP2Cβ (PPM1B) traces back to the 1980s [8,9]. The prototype 42 and 45 kDa proteins share 75% amino acid sequence identity [10]. They are the most abundant and best-studied members of all PP2C isoenzymes. In the meantime, two splice variants were found for the α-isoenzyme (36 and 42 kDa) and six splice variants for 2Cβ (42–55 kDa). PP2C enzymes are touching a variety of substrate classes, e.g. kinases, receptors, channels and transcription factors, thereby affecting quite diverse physiological effects, e.g. stress response, metabolism and cell cycle. By far the most is known about the substrates of PP2Cα and PP2Cβ; however, the other 2C isoenzymes are increasingly coming into play. The present knowledge of substrates of PP2C is summarized in Table 1 [1147]. A review on PP2C is not available in the literature.

Table 1
Substrates of eukaryotic PP2C isoenzymes

CaMKP (-N), CaMK phosphatase (nuclear); FIN 13, fibroblast growth factor-inducible protein 13; ILKAP, ILK1-associated phosphatase; n.d., not defined; NERPP, neuronal protein phosphatase; PHLPP, PH-domain leucine-rich repeat phosphatase; POPX, partner of PIX (PAK-interacting exchange factor); PrP-2C, recombinant rat PP2C; SCOP, suprachiasmatic nucleus circadian oscillatory protein; WIP1, wild-type p53-induced phosphatase.

Substrate  PP2C isoenzyme Reference 
AMPK 5′-AMP-activated protein kinase α (PPM1A) [11
ARC Apoptosis repressor with a caspase recruitment domain γ (PPM1F, FIN 13) [12
ASK1 Apoptosis signal-regulating kinase ϵ (PPM1L) [13
Axin Axis inhibition protein 1 α (PPM1A) [14
Bad  α (PPM1A), β (PPM1B) [15
Ca2+ channel  α (PPM1A) [16
CaMKI Ca2+/calmodulin-dependent protein kinase I CaMKP [17
CaMKII  PrP-2C (PPM1F), CaMKP [17,18
Nuclear CaMKII Nuclear CaMKII CaMKP-N [17
CaMKIV  CaMKP-N [17
CDK2 Cyclin-dependent kinase 2 α (PPM1A), β (PPM1B) [19
CDK6  α (PPM1A), β (PPM1B) [19
CFTR Cystic fibrosis transmembrane conductance regulator n.d. [20
CNS inhibitors Central nervous system inhibitors NERPP-2C [21
CSAD Cysteine sulfinic acid decarboxylase n.d. [22
DARP-32 Dopamine/cAMP-regulated phosphoprotein, 32 kDa n.d. [23
mGluR3 Metabotropic glutamate receptor 3 α (PPM1A), β (PPM1B) [24
   γ (PPM1G, FIN 13), δ2 (WIP1)  
Glycogen phosphorylase  n.d. [25
Glycogen synthase  n.d. [25
HMG-CoA-reductase 3-Hydroxy-3-methylglutaryl-coA-reductase α (PPM1A), β (PPM1B) [26
HMG-CoA-reductase-kinase HMG-CoA-reductase-kinase n.d. [27
HSL Hormone-sensitive lipase n.d. [28
IKKβ IκB (inhibitory κB) kinase complex β β (PPM1B) [29
ILK1 Integrin-linked kinase 1 ILKAP (δ1) [30
MKKs 3b, 4, 6 and 7 MAPK (mitogen-activated protein kinase) kinases 3b, 4, 6 and 7 α (PPM1A), β (PPM1B) [31
Moesin Membrane-organizing extension spike protein n.d. [32
NPR-A Natriuretic peptide receptor-A α (PPM1A), β (PPM1B) [33
p38 MAPK p38α α (PPM1A), δ2 (WIP1) [34,35
p53  α (PPM1A), δ2 (WIP1) [35,36
PAK p21-activated kinase POPX1, POPX2 [37
PKB/Akt Protein kinase B PHLPP1 (SCOPj[38
PKC Protein kinase C PHLPP1 (SCOP) [38
Phospholamban  n.d. [39
Phosphorylase kinase  n.d. [25
PR Progesterone receptor δ2 (WIP1) [40
RSK2 p90 ribosomal S6 kinase 2 δ2 (WIP1) [41
SEK1 SAPK (stress-activated protein kinase)/ERK α (PPM1A) [34
  (extracellular-signal-regulated kinase) kinase 1   
Smad  α (PPM1A) [42
TAK1 TGF-β (transforming growth factor-β)-activated kinase 1 β (PPM1B), ϵ (PPM1L) [43,44
TH Tyrosine hydroxylase α (PPM1A), β (PPM1B) [45
VASP Vasodilator-stimulated phosphoprotein n.d. [46
WASP Wiskott–Aldrich syndrome protein δ2 (WIP1) [47
Substrate  PP2C isoenzyme Reference 
AMPK 5′-AMP-activated protein kinase α (PPM1A) [11
ARC Apoptosis repressor with a caspase recruitment domain γ (PPM1F, FIN 13) [12
ASK1 Apoptosis signal-regulating kinase ϵ (PPM1L) [13
Axin Axis inhibition protein 1 α (PPM1A) [14
Bad  α (PPM1A), β (PPM1B) [15
Ca2+ channel  α (PPM1A) [16
CaMKI Ca2+/calmodulin-dependent protein kinase I CaMKP [17
CaMKII  PrP-2C (PPM1F), CaMKP [17,18
Nuclear CaMKII Nuclear CaMKII CaMKP-N [17
CaMKIV  CaMKP-N [17
CDK2 Cyclin-dependent kinase 2 α (PPM1A), β (PPM1B) [19
CDK6  α (PPM1A), β (PPM1B) [19
CFTR Cystic fibrosis transmembrane conductance regulator n.d. [20
CNS inhibitors Central nervous system inhibitors NERPP-2C [21
CSAD Cysteine sulfinic acid decarboxylase n.d. [22
DARP-32 Dopamine/cAMP-regulated phosphoprotein, 32 kDa n.d. [23
mGluR3 Metabotropic glutamate receptor 3 α (PPM1A), β (PPM1B) [24
   γ (PPM1G, FIN 13), δ2 (WIP1)  
Glycogen phosphorylase  n.d. [25
Glycogen synthase  n.d. [25
HMG-CoA-reductase 3-Hydroxy-3-methylglutaryl-coA-reductase α (PPM1A), β (PPM1B) [26
HMG-CoA-reductase-kinase HMG-CoA-reductase-kinase n.d. [27
HSL Hormone-sensitive lipase n.d. [28
IKKβ IκB (inhibitory κB) kinase complex β β (PPM1B) [29
ILK1 Integrin-linked kinase 1 ILKAP (δ1) [30
MKKs 3b, 4, 6 and 7 MAPK (mitogen-activated protein kinase) kinases 3b, 4, 6 and 7 α (PPM1A), β (PPM1B) [31
Moesin Membrane-organizing extension spike protein n.d. [32
NPR-A Natriuretic peptide receptor-A α (PPM1A), β (PPM1B) [33
p38 MAPK p38α α (PPM1A), δ2 (WIP1) [34,35
p53  α (PPM1A), δ2 (WIP1) [35,36
PAK p21-activated kinase POPX1, POPX2 [37
PKB/Akt Protein kinase B PHLPP1 (SCOPj[38
PKC Protein kinase C PHLPP1 (SCOP) [38
Phospholamban  n.d. [39
Phosphorylase kinase  n.d. [25
PR Progesterone receptor δ2 (WIP1) [40
RSK2 p90 ribosomal S6 kinase 2 δ2 (WIP1) [41
SEK1 SAPK (stress-activated protein kinase)/ERK α (PPM1A) [34
  (extracellular-signal-regulated kinase) kinase 1   
Smad  α (PPM1A) [42
TAK1 TGF-β (transforming growth factor-β)-activated kinase 1 β (PPM1B), ϵ (PPM1L) [43,44
TH Tyrosine hydroxylase α (PPM1A), β (PPM1B) [45
VASP Vasodilator-stimulated phosphoprotein n.d. [46
WASP Wiskott–Aldrich syndrome protein δ2 (WIP1) [47

32P-labelled casein is the classical substrate for determination of PP2C activity in vitro [48,49]. Using such an assay does not allow us to differentiate among 2C isoenzymes. Phosphopeptides can also be used as substrates; however, the behaviour of PP2C is different, e.g. altered bivalent cation dependence of PP2Cα and PP2Cβ [50]. Whether or not PP2Cα and PP2Cβ might act on a certain phosphoprotein is predictable to some extent [51]. Interestingly, PP2Cα and PP2Cβ are more active on phosphothreonyl peptides as compared with their physiological, otherwise identical, counterparts. An explanation on a molecular level is still missing.

Except for chelating Mg2+ and Mn2+ cations by EGTA or addition of Ca2+ ions at a high concentration, no reagent is known to inhibit the activity of PP2Cα and PP2Cβ. The inhibitors of serine/threonine protein phosphatases such as okadaic acid, microcystin, tautomycin or inhibitor proteins I1 and I2 have no effect on PP2C isoenzymes. Knockout mouse models are not available. At present, no reagent is available to specifically inhibit any of the PP2C family members. Searching for such a tool remains a challenge. One is left, at present, with RNAi (RNA interference). This option has proved successful [29,52].

Surprisingly, little is known about the regulation of PP2Cα and PP2Cβ. (i) Regulatory or targeting subunits have not been identified. (ii) The α isoenzyme was reported to undergo phosphorylation by protein kinase CK2 [53]. Nothing is known about the reverse reaction. (iii) Long ago, dimerization was suggested [54]. Although this had been neither proved incorrect nor verified, PP2Cα and PP2Cβ are described in the literature as monomeric proteins. (iv) Inhibition by Ca2+ ions was observed in several cases. However, the concentration applied was considered too high to account for physiological relevance [55,56]. This accumulation of unsolved problems (plus the lack of an inhibitor) are the major reasons for PP2C being neglected among the otherwise soaring development of protein phosphatase research.

Activation of PP2Cα and PP2Cβ by fatty acids

For more than 20 years, the activity of PP2Cα and PP2Cβ was determined in the presence of very high Mg2+ concentrations, 20 mM or more. It is now known that the same specific activity can be achieved at a physiological concentration of 0.5–1.5 mM Mg2+ provided unsaturated long-chain fatty acids are present [49]. The activities of PP2Cα and PP2Cβ can be stimulated 10–15-fold by certain lipophilic compounds. The structural requirements for such activators comprise a minimum chain length of 15 C atoms, at least one double bond in a special position, and cis-configuration. The carboxy group is crucial as well. Overall, PP2Cα and PP2Cβ revealed a most remarkable selectivity for the chemistry of their activators.

Specificity was also observed on the enzyme and substrate levels. The formation of unsaturated fatty acid esters allows us to differentiate the activation concerning PP2Cα and PP2Cβ (esters do not activate) compared with PP5 (esters stimulate to the same extent as the unbound acid). Activation of PP2Cα and PP2Cβ requires the integrity of a protein substrate [e.g. casein and BAD (Bcl-2/Bcl-XL-antagonist, causing cell death)]; it is not detectable with phosphopeptides [50].

PP2Cα and PP2Cβ are both sensitive to stimulation by unsaturated fatty acids. The extent of activation is slightly more expressed in the α isoform. There is evidence, however, that not all members of the PP2C family are susceptible to activity changes induced by unsaturated fatty acids (M. Dworak, S. Klumpp and J. Krieglstein, unpublished work). This is not surprising considering that PP2C-like enzymes from plants, e.g. MP2C from Medicago sativa, are even inhibited by unsaturated fatty acids [57]. On the other hand, the type-2C phosphatases from plants, e.g. ABI1 from Arabidopsis thaliana, are dependent on Mg2+ ions for activity. The C-terminal domain of the protein is related to PP2Cα and PP2Cβ, whereas the N-terminal extension contains an EF-hand calcium-binding site [58]. So far, nothing is known about the effect of unsaturated fatty acids on PP2Cs from bacteria or on the homologue identified in archaea.

Degeneration of neurons by PP2Cα and PP2Cβ

The phosphorylation state of the pro-apoptotic protein BAD contributes to cell survival [59]. Dephosphorylation of phospho-Ser112, -Ser136 and -Ser155 in BAD was shown to induce cell death. In the meantime, the number of phosphorylation sites within the 18 kDa BAD protein has reached seven and they are somewhat clustered within the C-terminal moiety of the protein. Many kinases and phosphatases are involved so that the hierarchy has not been resolved yet.

PP2Cα and PP2Cβ are capable of dephosphorylating at least four of the phosphorylation sites of BAD, including phospho-Thr117 [60]. Dephosphorylation of BAD by PP2Cα and PP2Cβ can be stimulated by lipophilic compounds as described above, e.g. oleic acid [61]. To test whether PP2Cα and PP2Cβ are involved in triggering the death machinery in vivo, we started working on cell-culture systems. Knowing about the presence and co-localization of BAD with PP2Cα and PP2Cβ in neurons, we focused on the cell line SH-SY5Y and on primary cultures prepared from the cerebral cortex or hippocampus of embryonic and postnatal rats respectively. The results obtained essentially were the same: addition of lipophilic compounds capable of stimulating PP2Cα and PP2Cβ activity resulted in cell death. This was detected microscopically by monitoring changes in morphology and counting condensed chromatin after staining of the nuclei with Hoechst 33258. In contrast, lipophilic reagents not effecting PP2Cα and PP2Cβ were not harmful to the cells. Staining with the dye Nile Blue was used to monitor uptake of the lipophilic compounds into the cells (Figure 1). These control experiments revealed that the difference observed (whether cell death occurred or not) was not due to different loading of the cells with lipids, and thus could be attributed to the chemistry of the lipids instead.

Uptake of fatty acids

Figure 1
Uptake of fatty acids

Uptake of oleic acid, eladaic acid and oleic acid methyl ester in HUVECs as demonstrated by staining with Nile Blue. HUVECs were incubated with vehicle (control), oleic acid, its trans derivative eladaic acid, and oleic acid methyl ester for 3 h at a concentration of 200 μM respectively. After 24 h, HUVECs treated with oleic acid showed apoptotic morphology, whereas HUVECs treated with eladaic acid or oleic acid methyl ester did not. Apoptotic damage was revealed after staining with Hoechst 33258.

Figure 1
Uptake of fatty acids

Uptake of oleic acid, eladaic acid and oleic acid methyl ester in HUVECs as demonstrated by staining with Nile Blue. HUVECs were incubated with vehicle (control), oleic acid, its trans derivative eladaic acid, and oleic acid methyl ester for 3 h at a concentration of 200 μM respectively. After 24 h, HUVECs treated with oleic acid showed apoptotic morphology, whereas HUVECs treated with eladaic acid or oleic acid methyl ester did not. Apoptotic damage was revealed after staining with Hoechst 33258.

This correlation, activation of PP2Cα and PP2Cβ with the induction of apoptosis, was studied in more detail using RNAi technology. Cells harbouring an artificially reduced amount of PP2Cα and PP2Cβ protein were significantly less susceptible to cell death by unsaturated fatty acids [29]. This is evidence that what had been considered a striking correlation at first glance finally extends to a causal relationship between activation of PP2Cα and PP2Cβ with apoptosis.

The nature of fatty acids and their potency to stimulate PP2Cα and PP2Cβ are crucial, whereas BAD might be just one of several substrates of PP2Cα and PP2Cβ in cell death signalling. Indeed, PP2Cα and PP2Cβ have been shown to be involved in p53 signalling pathways [36] and in stress-activated protein kinase cascades [62]. The link from PP2C to stress response obviously had been well conserved throughout evolution. PP2C was shown to regulate stress response in fission yeast [63] and to be involved in stress response the plant kingdom [64].

Endothelial apoptosis by PP2Cα and PP2Cβ

We started working on HUVECs (human umbilical-vein endothelial cells) mainly for two reasons. (i) To check whether the causal link between PP2Cα and PP2Cβ and cell death discovered in neurons is restricted to those cells or are possibly of broad relevance. (ii) In physiological terms, it is quite unlikely that neurons in vivo might meet the fairly high concentration of unsaturated fatty acids required for activation of PP2Cα and PP2Cβ. In contrast, endothelial cells are known to encounter massive amounts of fatty acids. This occurs after cleavage of lipoproteins and uptake of the fatty acids released. Under pathological conditions and excessive lipoprotein content, the locally achieved unbound (‘free’) fatty acid concentration persists at a high level.

Enzyme activity determination revealed the presence of PP2C in HUVECs and in macrophages. Immunocytochemistry not only demonstrated the presence of BAD in those cells but also showed co-localization of BAD with PP2Cα and PP2Cβ in the cytosol of HUVECs (Figure 2) and macrophages. This was the minimum prerequisite for dealing with a physiologically meaningful response. We were left with the question of whether unsaturated fatty acids as they are found in the human body might activate PP2Cα and PP2Cβ, thus possibly triggering cell death. For that purpose, we studied the effect of lipoproteins isolated from human blood [65]. VLDL (very-low-density lipoprotein), LDL (low-density lipoprotein) and HDL (high-density lipoprotein) neither activated PP2Cα and PP2Cβ nor were those lipoproteins harmful to the cells. Lipoproteins VLDL, LDL and HDL in the presence of a lipoprotein lipase, however, did activate PP2Cα and PP2Cβ. Concomitantly, cell death was observed. This can be explained by activation of PP2Cα and PP2Cβ by unsaturated fatty acids liberated from the lipoproteins by lipase treatment. Indeed, HPLC technology revealed oleic acid and linoleic acid as the major constituents of unsaturated fatty acids from lipoproteins. This may represent a mechanism of atherogenesis: unsaturated fatty acids from lipoproteins activate PP2Cα and PP2Cβ, thereby inducing apoptosis of endothelial cells. Indeed, endothelial dysfunction is considered to be an initial step in the development of atherosclerosis.

Co-localization of PP2Cβ and BAD in endothelial cells

Figure 2
Co-localization of PP2Cβ and BAD in endothelial cells

Fluorescence laser scanning photographs showing subcellular co-localization of BAD and PP2Cβ. (A) Incubation with the antibodies against PP2Cβ (1:100), and (B) antibodies directed against BAD (1:350). (C) Merge of (A) and (B). (D) Controls, omitting primary antibodies against PP2Cβ (top) or BAD (bottom) respectively. Scale bar, 10 μm.

Figure 2
Co-localization of PP2Cβ and BAD in endothelial cells

Fluorescence laser scanning photographs showing subcellular co-localization of BAD and PP2Cβ. (A) Incubation with the antibodies against PP2Cβ (1:100), and (B) antibodies directed against BAD (1:350). (C) Merge of (A) and (B). (D) Controls, omitting primary antibodies against PP2Cβ (top) or BAD (bottom) respectively. Scale bar, 10 μm.

Conclusion

Our studies of PP2Cα and PP2Cβ within the field of neurodegeneration and neuroprotection have taken an unexpected turn. The concentrations of unsaturated fatty acids required to activate PP2Cα and PP2Cβ and necessary for the induction of cell death directed us to endothelial cells and into the field of atherosclerosis.

PP2C enzymes had been neglected and treated as ‘black sheep’ phosphatases for many years mainly because nothing was known about activators and inhibitors, except for the dependence on Mg2+ ions. The inhibitor site still has not been resolved, although people currently are trying hard to find it [66]. Now that we know about the activators (unsaturated fatty acids) and have learned about the consequences (endothelial apoptosis), this may finally put PP2Cα and PP2Cβ back on stage as an interesting target to possibly prevent and treat atherosclerosis.

International Symposium on Neurodegeneration and Neuroprotection: Independent Meeting held at University of Münster, Germany, 23–27 July 2006. Organized and Edited by S. Klumpp and J. Krieglstein (Münster, Germany).

Abbreviations

     
  • BAD

    Bcl-2/Bcl-XL-antagonist, causing cell death

  •  
  • HDL

    high-density lipoprotein

  •  
  • HUVEC

    human umbilical-vein endothelial cell

  •  
  • ILK1

    integrin-linked kinase 1

  •  
  • LDL

    low-density lipoprotein

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • PAK

    p21-activated kinase

  •  
  • PP2Cα

    protein phosphatase type 2Cα

  •  
  • PPM

    protein phosphatase magnesium-dependent

  •  
  • RNAi

    RNA interference

  •  
  • VLDL

    very-low-density lipoprotein

The Deutsche Forschungsgemeinschaft supports work in our laboratory.

References

References
1
Krebs
E.G.
Beavo
J.A.
Annu. Rev. Biochem.
1979
, vol. 
48
 (pg. 
923
-
959
)
2
Hunter
T.
Cell
1995
, vol. 
80
 (pg. 
225
-
236
)
3
Cohen
P.
Eur. J. Biochem.
2001
, vol. 
268
 (pg. 
5001
-
5010
)
4
Ingebritsen
T.S.
Cohen
P.
Science
1983
, vol. 
221
 (pg. 
331
-
338
)
5
Cohen
P.T.
Trends Biochem Sci.
1997
, vol. 
22
 (pg. 
245
-
251
)
6
Barford
D.
Trends Biochem Sci.
1996
, vol. 
21
 (pg. 
407
-
412
)
7
Bork
P.
Brown
N.P.
Hegyi
H.
Schultz
J.
Protein Sci.
1996
, vol. 
5
 (pg. 
1421
-
1425
)
8
Hiraga
A.
Kikuchi
K.
Tamura
S.
Tsuiki
S.
Eur. J. Biochem.
1981
, vol. 
119
 (pg. 
503
-
510
)
9
Pato
M.D.
Adelstein
R.S.
J. Biol. Chem.
1983
, vol. 
258
 (pg. 
7055
-
7058
)
10
Wenk
J.
Trompeter
H.I.
Pettrich
K.G.
Cohen
P.T.
Campbell
D.G.
Mieskes
G.
FEBS Lett.
1992
, vol. 
297
 (pg. 
135
-
138
)
11
Moore
F.
Weekes
J.
Hardie
D.G.
Eur. J. Biochem.
1991
, vol. 
199
 (pg. 
691
-
697
)
12
Zhang
Y.Q.
Herman
B.
J. Cell Biochem.
2006
, vol. 
99
 (pg. 
575
-
588
)
13
Tamura
S.
Shinnosuke
T.
Saito
J.I.
Awano
K.
Kudo
T.A.
Kobayashi
T.
Cancer Sci.
2006
, vol. 
97
 (pg. 
563
-
567
)
14
Strovel
E.T.
Wu
D.
Sussman
D.J.
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
2399
-
2403
)
15
Klumpp
S.
Selke
D.
Krieglstein
J.
Neurochem. Int.
2003
, vol. 
42
 (pg. 
555
-
560
)
16
Li
D.
Wang
F.
Lai
M.
Chen
Y.
Zhang
J.F.
J. Neurosci.
2005
, vol. 
25
 (pg. 
1914
-
1923
)
17
Takeuchi
M.
Ishida
A.
Kameshita
I.
Kitani
T.
Okuno
S.
Fujisawa
H.
J. Biochem. (Tokyo)
2001
, vol. 
130
 (pg. 
833
-
840
)
18
Fukunaga
K.
Kobayashi
T.
Tamura
S.
Miyamoto
E.
J. Biol. Chem.
1993
, vol. 
268
 (pg. 
133
-
137
)
19
Cheng
A.
Kaldis
P.
Solomon
M.J.
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
34744
-
34749
)
20
Travis
S.M.
Berger
H.A.
Welsh
M.J.
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
11055
-
11060
)
21
Labes
M.
Roder
J.
Roach
A.
Mol. Cell. Neurosci.
1998
, vol. 
12
 (pg. 
29
-
47
)
22
Tang
X.W.
Hsu
C.C.
Schloss
J.V.
Faiman
M.D.
Wu
E.
Yang
C.Y.
Wu
J.Y.
J. Neurosci.
1997
, vol. 
17
 (pg. 
6947
-
6951
)
23
Desdouits
F.
Siciliano
J.C.
Nairn
A.C.
Greengard
P.
Girault
J.A.
Biochem. J.
1998
, vol. 
330
 (pg. 
211
-
216
)
24
Flajolet
M.
Rakhilin
S.
Wang
H.
Starkova
N.
Nuangchamnong
N.
Nairn
A.C.
Greengard
P.
Proc. Natl. Acad. Sci. U.S.A.
2003
, vol. 
100
 (pg. 
16006
-
16011
)
25
Ingebritsen
T.S.
Foulkes
J.G.
Cohen
P.
Eur. J. Biochem.
1983
, vol. 
132
 (pg. 
263
-
274
)
26
Ching
Y.P.
Kobayashi
T.
Tamura
S.
Hardie
D.G.
FEBS Lett.
1997
, vol. 
411
 (pg. 
265
-
268
)
27
Ingebritsen
T.S.
Cohen
P.
Eur. J. Biochem.
1983
, vol. 
132
 (pg. 
255
-
261
)
28
Olsson
H.
Belfrage
P.
Eur. J. Biochem.
1987
, vol. 
168
 (pg. 
399
-
405
)
29
Prajapati
S.
Verma
U.
Yamamoto
Y.
Kwak
Y.T.
Gaynor
R.B.
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
1739
-
1746
)
30
Leung-Hagesteijn
C.
Mahendra
A.
Naruszewicz
I.
Hannigan
G.E.
EMBO J.
2001
, vol. 
20
 (pg. 
2160
-
2170
)
31
Hanada
M.
Kobayashi
T.
Ohnishi
M.
Ikeda
S.
Wang
H.
Katsura
K.
Yanagawa
Y.
Hiraga
A.
Kanamaru
R.
Tamura
S.
FEBS Lett.
1998
, vol. 
437
 (pg. 
172
-
176
)
32
Hishiya
A.
Ohnishi
M.
Tamura
S.
Nakamura
F.
J. Biol. Chem.
1999
, vol. 
274
 (pg. 
26705
-
26712
)
33
Bryan
P.M.
Potter
L.R.
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
16041
-
16047
)
34
Takekawa
M.
Maeda
T.
Saito
H.
EMBO J.
1998
, vol. 
17
 (pg. 
4744
-
4752
)
35
Fiscella
M.
Zhang
H.
Fan
S.
Sakaguchi
K.
Shen
S.
Mercer
W.E.
Vande Woude
G.F.
O'Connor
P.M.
Apella
E.
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
6048
-
6053
)
36
Ofek
P.
Ben-Meir
D.
Kariv-Inbal
Z.
Oren
M.
Lavi
S.
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
14299
-
14305
)
37
Koh
C.G.
Tan
E.J.
Manser
E.
Lim
L.
Curr. Biol.
2002
, vol. 
12
 (pg. 
317
-
321
)
38
Gao
T.
Furnari
F.
Newton
A.C.
Mol. Cell
2005
, vol. 
18
 (pg. 
13
-
24
)
39
MacDougall
L.K.
Jones
L.R.
Cohen
P.
Eur. J. Biochem.
1991
, vol. 
196
 (pg. 
725
-
734
)
40
Proia
D.A.
Nannenga
B.W.
Donehower
L.A.
Weigel
N.L.
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
7089
-
7101
)
41
Doehn
U.
Gammeltoft
S.
Shen
S.H.
Jensen
C.J.
Biochem. J.
2004
, vol. 
382
 (pg. 
425
-
431
)
42
Lin
X.
Duan
X.
Liang
Y.Y.
Su
Y.
Wrighton
K.H.
Long
J.
Hu
M.
Davis
C.M.
Wang
J.
Brunicardi
F.C.
, et al. 
Cell
2006
, vol. 
125
 (pg. 
915
-
928
)
43
Hanada
M.
Ninomiya-Tsuji
J.
Komaki
K.
Ohnishi
M.
Katsura
K.
Kanamaru
R.
Matsumoto
K.
Tamura
S.
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
5753
-
5759
)
44
Li
M.G.
Katsura
K.
Nomiyama
H.
Komaki
K.
Ninomiya-Tsuji
J.
Matsumoto
K.
Kobayashi
T.
Tamura
S.
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
12013
-
12021
)
45
Bevilaqua
L.R.
Cammarota
M.
Dickson
P.W.
Sim
A.T.
Dunkley
P.R.
J. Neurochem.
2003
, vol. 
85
 (pg. 
1368
-
1373
)
46
Abel
K.
Mieskes
G.
Walter
U.
FEBS Lett.
1995
, vol. 
370
 (pg. 
184
-
188
)
47
Ramesh
N.
Anton
I.M.
Hartwig
J.H.
Geha
R.S.
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
14671
-
14676
)
48
McGowan
C.H.
Cohen
P.
Methods Enzymol.
1988
, vol. 
159
 (pg. 
416
-
426
)
49
Klumpp
S.
Selke
D.
Hermesmeier
J.
FEBS Lett.
1998
, vol. 
437
 (pg. 
229
-
232
)
50
Krieglstein
J.
Selke
D.
Maassen
A.
Klumpp
S.
Methods Enzymol.
2003
, vol. 
366
 (pg. 
282
-
289
)
51
Pinna
L.A.
Donella-Deana
A.
Biochim. Biophys. Acta.
1994
, vol. 
1222
 (pg. 
415
-
431
)
52
Schwarz
S.
Hufnagel
B.
Dworak
M.
Klumpp
S.
Krieglstein
J.
Apoptosis
2006
, vol. 
11
 (pg. 
1111
-
1119
)
53
Kobayashi
T.
Kanno
S.
Terasawa
T.
Murakami
T.
Ohnishi
M.
Ohtsuki
K.
Hiraga
A.
Tamura
S.
Biochem. Biophys. Res. Commun.
1993
, vol. 
195
 (pg. 
484
-
489
)
54
Mieskes
G.
Söling
H.D.
FEBS Lett.
1985
, vol. 
181
 (pg. 
7
-
11
)
55
Pato
M.D.
Kerc
E.
Mol. Cell. Biochem.
1991
, vol. 
101
 (pg. 
31
-
41
)
56
Wang
Y.
Santini
F.
Qin
K.
Huang
C.Y.
J. Biol. Chem.
1995
, vol. 
270
 (pg. 
25607
-
25612
)
57
Baudouin
E.
Meskiene
I.
Hirt
H.
Plant J.
1999
, vol. 
20
 (pg. 
343
-
348
)
58
Leube
M.P.
Grill
E.
Amrhein
N.
FEBS Lett.
1998
, vol. 
424
 (pg. 
100
-
104
)
59
Zha
J.
Harada
H.
Yang
E.
Jockel
J.
Korsmeyer
S.J.
Cell
1996
, vol. 
87
 (pg. 
619
-
628
)
60
Klumpp
S.
Mäurer
A.
Zhu
Y.
Aichele
D.
Pinna
L.A.
Krieglstein
J.
Neurochem. Int.
2004
, vol. 
45
 (pg. 
747
-
752
)
61
Klumpp
S.
Selke
D.
Ahlemeyer
B.
Schaper
C.
Krieglstein
J.
Neurochem. Int.
2002
, vol. 
41
 (pg. 
251
-
259
)
62
Tamura
S.
Hanada
M.
Ohnishi
M.
Katsura
K.
Sasaki
M.
Kobayashi
T.
Eur. J. Biochem.
2002
, vol. 
269
 (pg. 
1060
-
1066
)
63
Gaits
F.
Shiozaki
K.
Russell
P.
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
17873
-
17879
)
64
Meskiene
I.
Bogre
L.
Glaser
W.
Balog
J.
Brandstötter
M.
Zwerger
K.
Ammerer
G.
Hirt
H.
Proc. Natl. Acad. Sci. U.S.A.
1998
, vol. 
95
 (pg. 
1938
-
1943
)
65
Hufnagel
B.
Dworak
M.
Soufi
M.
Mester
Z.
Zhu
Y.
Schaefer
J.R.
Klumpp
S.
Krieglstein
J.
Atherosclerosis
2005
, vol. 
180
 (pg. 
245
-
254
)
66
Rogers
J.P.
Beuscher
A.E.
Flajolet
M.
McAvoy
T.
Nairn
A.C.
Olson
A.J.
Greengard
P.
J. Med. Chem.
2006
, vol. 
49
 (pg. 
1658
-
1667
)