Advertisement

Nerve Growth Factor Protects against 6-Hydroxydopamine-induced Oxidative Stress by Increasing Expression of Heme Oxygenase-1 in a Phosphatidylinositol 3-Kinase-dependent Manner*

Open AccessPublished:February 10, 2003DOI:https://doi.org/10.1074/jbc.M209164200
      The survival signal elicited by the phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated with inactivation of pro-apoptotic proteins and attenuation of the general stress-induced increase in reactive oxygen species (ROS). However, the mechanisms by which this pathway regulates intracellular ROS levels remain largely unexplored. In this study, we demonstrate that nerve growth factor (NGF) prevents the accumulation of ROS in dopaminergic PC12 cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves PI3K/Akt-dependent induction of the stress response protein heme oxygenase-1 (HO-1). The effect of NGF was mimicked by induction of HO-1 expression with CoCl2; by treatment with bilirubin, an end product of heme catabolism; and by infection with a retroviral expression vector for human HO-1. The relevance of HO-1 in NGF-induced ROS reduction was further demonstrated by the evidence that cells treated with the HO-1 inhibitor tin-protoporphyrin or infected with a retroviral expression vector for antisense HO-1 exhibited enhanced ROS release in response to 6-OHDA, despite the presence of the neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1 mRNA and protein and partially reversed its protective effect against 6-OHDA-induced ROS release. By contrast, cells transfected with a membrane-targeted active version of Akt1 exhibited increased HO-1 expression, even in the absence of NGF, and displayed a greatly attenuated production of ROS and apoptosis in response to 6-OHDA. These observations indicate that the PI3K/Akt pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.
      High levels of reactive oxygen species (ROS)
      The abbreviations used are: ROS
      reactive oxygen species
      NGF
      nerve growth factor
      PI3K
      phosphatidylinositol 3-kinase
      HO
      heme oxygenase
      6-OHDA
      6-hydroxydopamine
      EGFP
      enhanced green fluorescent protein
      myr
      myristoylated
      HE
      hydroethidine
      PE
      phycoerythrin
      7-AAD
      7-aminoactinomycin D
      1The abbreviations used are: ROS
      reactive oxygen species
      NGF
      nerve growth factor
      PI3K
      phosphatidylinositol 3-kinase
      HO
      heme oxygenase
      6-OHDA
      6-hydroxydopamine
      EGFP
      enhanced green fluorescent protein
      myr
      myristoylated
      HE
      hydroethidine
      PE
      phycoerythrin
      7-AAD
      7-aminoactinomycin D
      induce cell death in the nervous system and have been associated with a number of pathologies such as Parkinson's disease (
      • Pettmann B.
      • Henderson C.E.
      ). Neurotrophins, including nerve growth factor (NGF), promote survival of target neurons and attenuate ROS-induced cell death (
      • Wiesmann C.
      • Ultsch M.H.
      • Bass S.H.
      • de Vos A.M.
      ). A prominent mechanism involved in NGF-induced cell survival consists of the activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effectors, including the protein kinase B/Akt family of Ser/Thr kinases (
      • Kaplan D.R.
      • Miller F.D.
      ,
      • Brazil D.P.
      • Hemmings B.A.
      ,
      • Burgering B.M.
      • Kops G.J.
      ). This pathway exerts protective actions against oxidative damage in central and peripheral neurons (
      • Brunet A.
      • Datta S.R.
      • Greenberg M.E.
      ). In this context, neuregulin prevents H2O2 induction of ROS in a PI3K-dependent manner (
      • Goldshmit Y.
      • Erlich S.
      • Pinkas-Kramarski R.
      ). Loss of oxidative stress tolerance with aging has been linked in part to reduced Akt kinase activity in old rats (
      • Ikeyama S.
      • Kokkonen G.
      • Shack S.
      • Wang X.T.
      • Holbrook N.J.
      ). Concerning neurodegeneration, we have recently reported the protective effect of active Akt1 against peptides of β-amyloid protein, characteristic of senile plaques found in the brains of Alzheimer's patients (
      • Martı́n D.
      • Salinas M.
      • Lopez-Valdaliso R.
      • Serrano E.
      • Recuero M.
      • Cuadrado A.
      ); against the Parkinson's disease-inducing toxin 1-methyl-4-phenylpyridinium (
      • Salinas M.
      • Martı́n D.
      • Alvarez A.
      • Cuadrado A.
      ); and against apoptotic concentrations of H2O2 (
      • Martı́n D.
      • Salinas M.
      • Fujita N.
      • Tsuruo T.
      • Cuadrado A.
      ). In these cases, expression of a constitutively active version of Akt prevented the increase in ROS that follows treatment of PC12 cells with these toxins.
      However, although those studies support an involvement of Akt in counteracting oxidative damage in neurons, a direct role of this pathway in the regulation of canonical antioxidant defenses, represented by the superoxide dismutases and/or the catalase/glutathione peroxidase system, has not been identified. On the contrary, recent reports on the dauer stage ofCaenorhabditis elegans and quiescent mammalian cells indicate that, in the absence of active PI3K and Akt, forkhead transcription factors (DAF-16 and FOXO3a, respectively) increase the expression of mitochondrial superoxide dismutase (
      • Honda Y.
      • Honda S.
      ,
      • Kops G.J.
      • Dansen T.B.
      • Polderman P.E.
      • Saarloos I.
      • Wirtz K.W.A.
      • Coffer P.J.
      • Huang T.T.
      • Bos J.L.
      • Medena R.H.
      • Burgering B.M.T.
      ). Moreover, mutations in the daf-2 network of C. elegans that inactivate PI3K and Akt lead to up-regulation of a cytosolic catalase (
      • Tabú J.
      • Lau J.F.
      • Ma C.
      • Hahn J.H.
      • Hoque R.
      • Rothblatt J.
      • Chalfie M.
      ); and in Drosophila melanogaster, inactivation of this pathway results in increased superoxide dismutase activity (
      • Clancy D.J.
      • Gems D.
      • Harshman L.G.
      • Oldham S.
      • Stocker H.
      • Hafen E.
      • Leevers S.J.
      • Partridge L.
      ). Therefore, activation of PI3K and Akt must lead to up-regulation of other ROS detoxification systems.
      In addition to the well characterized ROS scavenger systems mentioned above, emerging evidence supports a role for heme oxygenase (HO) enzymes as important components of the cellular antioxidant armamentarium (
      • Morse D.
      • Choi A.M.
      ,
      • Otterbein L.E.
      • Choi A.M.
      ). The heme oxygenase family is composed of at least two well characterized isoenzymes: inducible HO-1 and constitutive HO-2. HO-1, also known as HSP32 (heatshock protein of 32 kDa), is a stress response protein whose expression is induced in practically all tissues and cells tested in response to multiple oxidative insults such as heme, UV light, heavy metals, glutathione depletion, and H2O2. This enzyme catalyzes the stepwise degradation of heme to release free iron and equimolar concentrations of carbon monoxide (CO) and the linear tetrapyrrol biliverdin, which is converted to bilirubin by the enzyme biliverdin reductase. Many reports have established the potent antioxidant activity of biliverdin and bilirubin and the cytoprotective actions of CO on vascular endothelium and nerve cells (
      • Morse D.
      • Choi A.M.
      ,
      • Otterbein L.E.
      • Choi A.M.
      ,
      • Baranano D.E.
      • Snyder S.H.
      ,
      • Dennery P.A.
      ). Therefore, it is now widely accepted that induction of heme catabolism represents an adaptive, and ultimately protective, response to oxidative injury. Of particular relevance to this study, it has also been shown that the normally low levels of HO-1 expression in neurons (
      • Ewing J.F.
      • Haber S.N.
      • Maines M.D.
      ,
      • Takeda A.
      • Onodera H.
      • Sugimoto A.
      • Itoyama Y.
      • Kogure K.
      • Shibahara S.
      ) dramatically increase after formation of brain neurofibrillary tangles in Alzheimer's patients (
      • Takeda A.
      • Perry G.
      • Abraham N.G.
      • Dwyer B.E.
      • Kutty R.K.
      • Laitinen J.T.
      • Petersen R.B.
      • Smith M.A.
      ) and neuronal Lewy bodies in the substantia nigra of Parkinson's patients (
      • Schipper H.M.
      • Liberman A.
      • Stopa E.G.
      ) and that cerebellar granular neurons overexpressing HO-1 are resistant to glutamate-mediated oxidative stress (
      • Chen K.
      • Gunter K.
      • Maines M.D.
      ).
      The progressive deterioration of catecholaminergic cells in Parkinson's patients has been attributed, at least in part, to the high vulnerability of these cells to oxidative damage. One potential source of ROS in the substantia nigra includes the autoxidation of the neurotransmitter dopamine to generate 6-hydroxydopamine (6-OHDA) (
      • Blum D.
      • Torch S.
      • Lambeng N.
      • Nissou M.
      • Benabid A.L.
      • Sadoul R.
      • Verna J.M.
      ). In fact, 6-OHDA is present in rodent and human brains and has been widely used in experimental models of Parkinson's disease. Evidences derived from in vivo and in vitro experimental models of this disease have demonstrated that 6-OHDA neurotoxicity involves oxidative damage to catecholaminergic neurons (
      • Lotharius J.
      • Dugan L.L.
      • O'Malley K.L.
      ) via the generation of hydroxyl radicals, monoamine oxidase-mediated formation of H2O2, and mitochondrial generation of superoxide (
      • Blum D.
      • Torch S.
      • Lambeng N.
      • Nissou M.
      • Benabid A.L.
      • Sadoul R.
      • Verna J.M.
      ).
      With the aim to determine whether PI3K and Akt modulate the heme oxygenase system of ROS detoxification, we analyzed the effect of NGF on the regulation of HO-1 expression in catecholaminergic PC12 cells. We show that NGF-induced activation of PI3K/Akt up-regulates the expression and activity of HO-1, which, in turn, provides protection against 6-OHDA-induced oxidative damage in PC12 cells. These data contribute to revealing the mechanism whereby NGF and PI3K/Akt provide protection against oxidative injury and may be potentially relevant in the development of new therapies for neurodegenerative disorders such as Parkinson's disease.

      DISCUSSION

      This study documents a new physiological role for the PI3K/Akt survival pathway activated by the neurotrophic factor NGF: control of intracellular levels of oxygen free radicals by regulating the expression of HO-1. Previous studies have indicated that NGF elicits a protective effect against oxidative stress both in PC12 cells and in cultured neurons (
      • Wang W.
      • Post J.I.
      • Dow K.E.
      • Shin S.H.
      • Riopelle R.J.
      • Ross G.M.
      ,
      • Kirschner P.B.
      • Jenkins B.G.
      • Schulz J.B.
      • Finkelstein S.P.
      • Matthews R.T.
      • Rosen B.R.
      • Beal M.F.
      ). It has also been shown that NGF may modulate the canonical antioxidant machinery of ROS detoxification by increasing the expression of catalase and glutathione peroxidase activities (
      • Satoh T.
      • Yamagata T.
      • Ishikawa Y.
      • Yamada M.
      • Uchiyama Y.
      • Hatanaka H.
      ,
      • Sampath D.
      • Perez-Polo R.
      ). Our experiments further demonstrate that NGF regulates the expression of the novel antioxidant mechanism involving the stress protein HO-1. Moreover, enhanced HO-1 expression is essential for NGF function in ROS detoxification because cells expressing antisense HO-1 retroviral constructs were insensitive to NGF protection against 6-OHDA-induced oxidative stress. Although PI3K/Akt is a well documented pathway involved in protecting against apoptosis insults, including oxidative stress, this is the first report linking this survival pathway with a specific enzyme involved in ROS detoxification of mammalian cells. We show that the PI3K/Akt pathway is both necessary and sufficient for NGF-dependent abrogation of ROS levels in PC12 cells exposed to 6-OHDA.
      The protective role of the stress protein HO-1 in the brain has not been accepted until recently. Initially, post-mortem examination of human brain specimens revealed that expression of HO-1 immunoreactivity decorates Lewy bodies in the substantia nigra of Parkinson's patients (
      • Schipper H.M.
      • Liberman A.
      • Stopa E.G.
      ). This led to the assumption that HO-1 overactivity might not protect, but rather contribute to the development of parkinsonism because iron accumulation in dopaminergic neurons may contribute to chronic oxidative damage, leading to neurodegeneration in Parkinson's patients. A protective role for HO catabolism in the nervous system has been also challenged by the fact that hyperbilirubinemia is commonly associated with nerve cell injury and brain damage during severe neonatal jaundice and Crigler-Najjar type II syndrome (
      • Gourley G.R.
      ,
      • Rubboli G.
      • Ronchi F.
      • Cecchi P.
      • Rizzi R.
      • Gardella E.
      • Meletti S.
      • Zaniboni A.
      • Volpi L.
      • Tassinari C.A.
      ). Consistent with these observations, we could not obtain high constitutive levels of HO-1 expression either in HO-1-overexpressing cells (2.3-fold) or in myr-EGFP-Akt1 cells (2.5-fold). It is interesting, however, that short-term stimulation with NGF or other inducers of HO-1 such as hemin and CoCl2 may yield much higher transient levels of HO-1 expression. These observations are in agreement with the notion that HO-1 is a stress response protein (HSP32).
      Therefore, a key point toward establishing the protective or noxious effect on the HO system under conditions of oxidative injury may be related to the relative abundance of HO enzymes and their products. Low concentrations of bilirubin derived from HO-2 overexpression protect against neuronal oxidant injury (
      • Dore S.
      • Takahashi M.
      • Ferris C.D.
      • Zakhary R.
      • Hester L.D.
      • Guastella D.
      • Snyder S.H.
      ), and moderated levels of bilirubin exert antioxidant actions in the neonate and protect against retinopathy in premature newborns (
      • Hegyi T.
      • Goldie E.
      • Hiatt M.
      ). Likewise, recent reports have shown that transgenic mice overexpressing moderate levels of HO-1 display augmented resistance to neural damage in animal models of cerebral ischemia (
      • Panahian N.
      • Yoshiura M.
      • Maines M.D.
      ) or after H2O2- and glutamate-induced oxidant stress in vitro (
      • Chen K.
      • Gunter K.
      • Maines M.D.
      ). Accordingly, we have shown that constitutive but moderate overexpression of HO-1 attenuated 6-OHDA-induced ROS generation.
      The HO-1 gene contains a complex promoter with a large variety of regulatory elements (
      • Elbirt K.K.
      • Bonkovsky H.L.
      ). Some of these sequences might be putative candidates for regulation by PI3K and Akt. As a heat shock protein, the promoter of HO-1 contains several heat shock elements that may be negatively regulated by glycogen synthase kinase-3-mediated phosphorylation of heat shock factor-1. Because Akt phosphorylates and inhibits glycogen synthase kinase-3 (
      • Xavier I.J.
      • Mercier P.A.
      • McLoughlin C.M.
      • Ali A.
      • Woodgett J.R.
      • Ovsenek N.
      ), these might be sites of indirect regulation by this pathway. Another putative site involves the antioxidant-responsive element. During oxidative stress, the basic leucine zipper transcription factor Nrf2 heterodimerizes with small Maf to bind and activate antioxidant-responsive element sequences (
      • Kataoka K.
      • Handa H.
      • Nishizawa M.
      ). Although the putative regulation of Nrf2 by PI3K is poorly defined, recent reports suggest that PI3K regulates nuclear translocation of Nrf2 through actin rearrangement in response to oxidative stress (
      • Kang K.W.
      • Lee S.J.
      • Park J.W.
      • Kim S.G.
      ). We are currently analyzing these and other promoter regions that might ultimately respond to PI3K/Akt regulation.
      Given the variety of enzymes involved in oxidative detoxification, a striking finding of this study is the strong dependence on HO-1 to protect against 6-OHDA-induced oxidative stress. This may be explained by considering the mechanism involved in 6-OHDA toxicity. Probably, H2O2 is the most abundant form of ROS generated by 6-OHDA (
      • Blum D.
      • Torch S.
      • Lambeng N.
      • Nissou M.
      • Benabid A.L.
      • Sadoul R.
      • Verna J.M.
      ), and bilirubin is particularly well suited to deal with this compound because nanomolar amounts of bilirubin reduce micromolar amounts of H2O2 (
      • Dore S.
      • Takahashi M.
      • Ferris C.D.
      • Zakhary R.
      • Hester L.D.
      • Guastella D.
      • Snyder S.H.
      ). Moreover, other antioxidant enzymes may be regulated by by-products of HO-1 activity, thus contributing to ROS detoxification. For example, HO-1 activates the expression of mitochondrial superoxide dismutase in neonatal rat astroglia challenged with dopamine (
      • Frankel D.
      • Mehindate K.
      • Schipper H.M.
      ).
      Finally, we discovered that membrane-targeted active Akt prevented the effects of 6-OHDA not only on ROS production, but also on apoptosis, further suggesting that moderate overexpression of HO-1 through the PI3K/Akt survival pathway may be an important element for prevention of both phenomena. Considering the importance of apoptosis and ROS in neurodegeneration, this study suggests that activation of the PI3K/Akt pathway might have a therapeutic use in the treatment of oxidative stress-related neurodegenerative disorders such as Parkinson's disease.

      References

        • Pettmann B.
        • Henderson C.E.
        Neuron. 1998; 20: 633-647
        • Wiesmann C.
        • Ultsch M.H.
        • Bass S.H.
        • de Vos A.M.
        Nature. 1999; 401: 184-188
        • Kaplan D.R.
        • Miller F.D.
        Curr. Opin. Neurobiol. 2000; 10: 381-391
        • Brazil D.P.
        • Hemmings B.A.
        Trends Biochem. Sci. 2001; 26: 657-664
        • Burgering B.M.
        • Kops G.J.
        Trends Biochem. Sci. 2002; 27: 352-360
        • Brunet A.
        • Datta S.R.
        • Greenberg M.E.
        Curr. Opin. Neurobiol. 2001; 11: 297-305
        • Goldshmit Y.
        • Erlich S.
        • Pinkas-Kramarski R.
        J. Biol. Chem. 2001; 276: 46379-46385
        • Ikeyama S.
        • Kokkonen G.
        • Shack S.
        • Wang X.T.
        • Holbrook N.J.
        FASEB J. 2002; 16: 114-116
        • Martı́n D.
        • Salinas M.
        • Lopez-Valdaliso R.
        • Serrano E.
        • Recuero M.
        • Cuadrado A.
        J. Neurochem. 2001; 78: 1000-1008
        • Salinas M.
        • Martı́n D.
        • Alvarez A.
        • Cuadrado A.
        Mol. Cell. Neurosci. 2001; 17: 67-77
        • Martı́n D.
        • Salinas M.
        • Fujita N.
        • Tsuruo T.
        • Cuadrado A.
        J. Biol. Chem. 2002; 277: 42943-42952
        • Honda Y.
        • Honda S.
        FASEB J. 1999; 13: 1385-1393
        • Kops G.J.
        • Dansen T.B.
        • Polderman P.E.
        • Saarloos I.
        • Wirtz K.W.A.
        • Coffer P.J.
        • Huang T.T.
        • Bos J.L.
        • Medena R.H.
        • Burgering B.M.T.
        Nature. 2002; 419: 316-321
        • Tabú J.
        • Lau J.F.
        • Ma C.
        • Hahn J.H.
        • Hoque R.
        • Rothblatt J.
        • Chalfie M.
        Nature. 1999; 399: 162-166
        • Clancy D.J.
        • Gems D.
        • Harshman L.G.
        • Oldham S.
        • Stocker H.
        • Hafen E.
        • Leevers S.J.
        • Partridge L.
        Science. 2001; 292: 104-106
        • Morse D.
        • Choi A.M.
        Am. J. Respir. Cell Mol. Biol. 2002; 27: 8-16
        • Otterbein L.E.
        • Choi A.M.
        Am. J. Physiol. 2000; 279: L1029-L1037
        • Baranano D.E.
        • Snyder S.H.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10996-11002
        • Dennery P.A.
        Curr. Top. Cell. Regul. 2000; 36: 181-199
        • Ewing J.F.
        • Haber S.N.
        • Maines M.D.
        J. Neurochem. 1992; 58: 1140-1149
        • Takeda A.
        • Onodera H.
        • Sugimoto A.
        • Itoyama Y.
        • Kogure K.
        • Shibahara S.
        Brain Res. 1994; 666: 120-124
        • Takeda A.
        • Perry G.
        • Abraham N.G.
        • Dwyer B.E.
        • Kutty R.K.
        • Laitinen J.T.
        • Petersen R.B.
        • Smith M.A.
        J. Biol. Chem. 2000; 275: 5395-5399
        • Schipper H.M.
        • Liberman A.
        • Stopa E.G.
        Exp. Neurol. 1998; 150: 60-68
        • Chen K.
        • Gunter K.
        • Maines M.D.
        J. Neurochem. 2000; 75: 304-313
        • Blum D.
        • Torch S.
        • Lambeng N.
        • Nissou M.
        • Benabid A.L.
        • Sadoul R.
        • Verna J.M.
        Prog. Neurobiol. 2001; 65: 135-172
        • Lotharius J.
        • Dugan L.L.
        • O'Malley K.L.
        J. Neurosci. 1999; 19: 1284-1293
        • Quan S.
        • Yang L.
        • Abraham N.G.
        • Kappas A.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12203-12208
        • Ryter S.W.
        • Kvam E.
        • Tyrrell R.M.
        Methods Mol. Biol. 2000; 99: 369-391
        • Chomczynski P.
        • Sacchi N.
        Anal. Biochem. 1987; 162: 156-159
        • Bindokas V.P.
        • Jordan J.
        • Lee C.C.
        • Miller R.J.
        J. Neurosci. 1996; 16: 1324-1336
        • Bindokas V.P.
        • Kuznetsov A.
        • Sreenan S.
        • Polonsky K.S.
        • Roe M.W.
        • Philipson L.H.
        J. Biol. Chem. 2003; 278: 9796-9801
        • Ohashi T.
        • Mizutani A.
        • Murakami A.
        • Kojo S.
        • Ishii T.
        • Taketani S.
        FEBS Lett. 2002; 511: 21-27
        • Chen K.
        • Maines M.D.
        Cell. Mol. Biol. 2000; 46: 609-617
        • Hill-Kapturczak N.
        • Truong L.
        • Thamilselvan V.
        • Visner G.A
        • Nick H.S.
        • Agarwal A.
        J. Biol. Chem. 2000; 275: 40904-40909
        • Llesuy S.F.
        • Tomaro M.L.
        Biochim. Biophys. Acta. 1994; 1223: 9-14
        • Li Volti G.
        • Wang J.
        • Traganos F.
        • Kappas A.
        • Abraham N.G.
        Biochem. Biophys. Res. Commun. 2002; 296: 1077-1082
        • Wang W.
        • Post J.I.
        • Dow K.E.
        • Shin S.H.
        • Riopelle R.J.
        • Ross G.M.
        Neurosci. Lett. 1999; 259: 115-118
        • Kirschner P.B.
        • Jenkins B.G.
        • Schulz J.B.
        • Finkelstein S.P.
        • Matthews R.T.
        • Rosen B.R.
        • Beal M.F.
        Brain Res. 1996; 713: 178-185
        • Satoh T.
        • Yamagata T.
        • Ishikawa Y.
        • Yamada M.
        • Uchiyama Y.
        • Hatanaka H.
        J. Biochem. (Tokyo). 1999; 125: 952-959
        • Sampath D.
        • Perez-Polo R.
        Neurochem. Res. 1997; 22: 351-362
        • Gourley G.R.
        Adv. Pediatr. 1997; 44: 173-229
        • Rubboli G.
        • Ronchi F.
        • Cecchi P.
        • Rizzi R.
        • Gardella E.
        • Meletti S.
        • Zaniboni A.
        • Volpi L.
        • Tassinari C.A.
        Neuropediatrics. 1997; 28: 281-286
        • Dore S.
        • Takahashi M.
        • Ferris C.D.
        • Zakhary R.
        • Hester L.D.
        • Guastella D.
        • Snyder S.H.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2445-2450
        • Hegyi T.
        • Goldie E.
        • Hiatt M.
        J. Perinatol. 1994; 14: 296-300
        • Panahian N.
        • Yoshiura M.
        • Maines M.D.
        J. Neurochem. 1999; 72: 1187-1203
        • Elbirt K.K.
        • Bonkovsky H.L.
        Proc. Assoc. Am. Physicians. 1999; 111: 438-447
        • Xavier I.J.
        • Mercier P.A.
        • McLoughlin C.M.
        • Ali A.
        • Woodgett J.R.
        • Ovsenek N.
        J. Biol. Chem. 2000; 275: 29147-29152
        • Kataoka K.
        • Handa H.
        • Nishizawa M.
        J. Biol. Chem. 2001; 276: 34074-34081
        • Kang K.W.
        • Lee S.J.
        • Park J.W.
        • Kim S.G.
        Mol. Pharmacol. 2002; 62: 1001-1010
        • Frankel D.
        • Mehindate K.
        • Schipper H.M.
        J. Cell. Physiol. 2000; 185: 80-86