Pam and Its Ortholog Highwire Interact with and May Negatively Regulate the TSC1·TSC2 Complex

Abstract

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder associated with mutations in TSC1, which codes for hamartin, or TSC2, which codes for tuberin. The brain is one of the most severely affected organs, and CNS lesions include cortical tubers and subependymal giant cell astrocytomas, resulting in mental retardation and seizures. Tuberin and hamartin function together as a complex in mammals and Drosophila. We report here the association of Pam, a protein identified as an interactor of Myc, with the tuberin-hamartin complex in the brain. The C terminus of Pam containing the RING zinc finger motif binds to tuberin. Pam is expressed in embryonic and adult brain as well as in cultured neurons. Pam has two forms in the rat CNS, an ∼450-kDa form expressed in early embryonic stages and an ∼350-kDa form observed in the postnatal period. In cortical neurons, Pam co-localizes with tuberin and hamartin in neurites and growth cones. Although Pam function(s) are yet to be defined, the highly conserved Pam homologs, HIW (Drosophila) and RPM-1 (Caenorhabditis elegans), are neuron-specific proteins that regulate synaptic growth. Here we show that HIW can genetically interact with the Tsc1·Tsc2 complex in Drosophila and could negatively regulate Tsc1·Tsc2 activity. Based on genetic studies, HIW has been implicated in ubiquitination, possibly functioning as an E3 ubiquitin ligase through the RING zinc finger domain. Therefore, we hypothesize that Pam, through its interaction with tuberin, could regulate the ubiquitination and proteasomal degradation of the tuberin-hamartin complex particularly in the CNS. Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder associated with mutations in TSC1, which codes for hamartin, or TSC2, which codes for tuberin. The brain is one of the most severely affected organs, and CNS lesions include cortical tubers and subependymal giant cell astrocytomas, resulting in mental retardation and seizures. Tuberin and hamartin function together as a complex in mammals and Drosophila. We report here the association of Pam, a protein identified as an interactor of Myc, with the tuberin-hamartin complex in the brain. The C terminus of Pam containing the RING zinc finger motif binds to tuberin. Pam is expressed in embryonic and adult brain as well as in cultured neurons. Pam has two forms in the rat CNS, an ∼450-kDa form expressed in early embryonic stages and an ∼350-kDa form observed in the postnatal period. In cortical neurons, Pam co-localizes with tuberin and hamartin in neurites and growth cones. Although Pam function(s) are yet to be defined, the highly conserved Pam homologs, HIW (Drosophila) and RPM-1 (Caenorhabditis elegans), are neuron-specific proteins that regulate synaptic growth. Here we show that HIW can genetically interact with the Tsc1·Tsc2 complex in Drosophila and could negatively regulate Tsc1·Tsc2 activity. Based on genetic studies, HIW has been implicated in ubiquitination, possibly functioning as an E3 ubiquitin ligase through the RING zinc finger domain. Therefore, we hypothesize that Pam, through its interaction with tuberin, could regulate the ubiquitination and proteasomal degradation of the tuberin-hamartin complex particularly in the CNS. Tuberous sclerosis complex (TSC) 1The abbreviations used are: TSC, tuberous sclerosis complex; ERM, ezrin, radixin, and moesin; CNS, central nervous system; HIW, highwire; RPM-1, regulator of presynaptic morphology-1; RZF, RING zinc finger; DIV, days in vitro; E3, ubiquitin-protein isopeptide ligase; S6K, S6 kinase; aa, amino acid(s); GST, glutathione S-transferase; d, Drosophila. is an autosomal dominant disorder characterized by benign hamartomas in brain, kidney, heart, lung, and skin. It is a developmental disorder with abnormalities in cell migration, differentiation, and proliferation. Neurological complications include seizures, mental retardation, and autism (1.Gomez M. Sampson J. Holtes-Whittemore V. Tuberous Sclerosi Complex. 3rd Ed. Oxford University Press, Oxford, United Kingdom1999Google Scholar). The disease is caused by mutations in tumor suppressor genes TSC1 or TSC2 encoding hamartin and tuberin, respectively (2.The European Chromosome 16 Tuberous Sclerosis ConsortiumCell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1511) Google Scholar, 3.van Slegtenhorst M. deHoogst R. Hermans C. Nellist M. Janssen B. Verhoef S. Lindhout D. et al.Science. 1997; 277: 805-808Crossref PubMed Scopus (1397) Google Scholar). Hamartin and tuberin associate in vivo forming a complex with other proteins (4.Nellist M. van Slegtenhorst M. Goedbloed M. van den Ouweland A.M. Halley D.J. van der Sluijs P. J. Biol. Chem. 1999; 274: 35647-35652Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Rodent models of Tsc1 and Tsc2 develop renal cyst adenomas with liver hemangiomas observed in mouse models (5.Kobayashi T. Minowa O. Kuno J. Mitani H. Hino O. Noda T. Cancer Res. 1999; 59: 1206-1211PubMed Google Scholar, 6.Onda H. Lueck A. Marks P.W. Warren H.B. Kwiatkowski D.J. J. Clin. Invest. 1999; 104: 687-695Crossref PubMed Scopus (322) Google Scholar, 7.Kobayashi T. Minowa O. Sugitani Y. Takai S. Mitani H. Kobayashi E. Noda T. Hino O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8762-8767Crossref PubMed Scopus (192) Google Scholar, 8.Kwiatkowski D.J. Zhang H. Bandura J.L. Heiberger K.M. Glogauer M. el-Hashemite N. Onda H. Hum. Mol. Genet. 2002; 11: 525-534Crossref PubMed Scopus (538) Google Scholar). Mutations in either Drosophila Tsc1 or Tsc2 show increase in cell and organ size, whereas co-expression of dTsc1 and dTsc2 inhibits growth and reduces cell size (9.Ito N. Rubin G. Cell. 1999; 96: 529-539Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 10.Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 11.Gao X. Pan D. Genes Dev. 2001; 15: 1383-1392Crossref PubMed Scopus (390) Google Scholar, 12.Tapon N. Ito N. Dickson B.J. Treisman J.E. Hariharan I.K. Cell. 2001; 105: 345-355Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Studies from fly also suggest that dS6 kinase may be an inhibitory target of dTsc1 and dTsc2 in growth control (10.Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 11.Gao X. Pan D. Genes Dev. 2001; 15: 1383-1392Crossref PubMed Scopus (390) Google Scholar, 12.Tapon N. Ito N. Dickson B.J. Treisman J.E. Hariharan I.K. Cell. 2001; 105: 345-355Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Recent studies have confirmed that mammalian TSC1 and TSC2 inhibit S6 kinase (S6K). Cells harboring mutations in either TSC1 or TSC2 have constitutively high phosphorylation of both S6K and its substrate S6 (8.Kwiatkowski D.J. Zhang H. Bandura J.L. Heiberger K.M. Glogauer M. el-Hashemite N. Onda H. Hum. Mol. Genet. 2002; 11: 525-534Crossref PubMed Scopus (538) Google Scholar, 13.Goncharova E.A. Goncharov D.A. Eszterhas A. Hunter D.S. Glassberg M.K. Yeung R.S. Walker C.L. Noonan D. Kwiatkowski D.J. Chou M.M. Panettieri Jr., R.A. Krymskaya V.P. J. Biol. Chem. 2002; 277: 30958-30967Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 14.Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Molecular Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar, 15.Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (575) Google Scholar, 16.Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 17.Jaeschke A. Hartkamp J. Saitoh M. Roworth W. Nobukuni T. Hodges A. Sampson J. Thomas G. Lamb R. J. Cell Biol. 2002; 159: 217-224Crossref PubMed Scopus (184) Google Scholar, 18.Kenerson H.L. Aicher L.D. True L.D. Yeung R.S. Cancer Res. 2002; 62: 5645-5650PubMed Google Scholar). Furthermore, tuberin and hamartin function together to inhibit target of rapamycin-mediated signaling to S6K in mammals as well as in fly (15.Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (575) Google Scholar, 16.Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 18.Kenerson H.L. Aicher L.D. True L.D. Yeung R.S. Cancer Res. 2002; 62: 5645-5650PubMed Google Scholar, 19.Tee A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13571-13576Crossref PubMed Scopus (697) Google Scholar). Akt phosphorylates tuberin and inhibits tuberin-hamartin function (14.Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Molecular Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar, 16.Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 20.Dan H.C. Sun M. Yang L. Feldman R.I. Sui X.M. Ou C.C. Nellist M. Yeung R.S. Halley D.J. Nicosia S.V. Pledger W.J. Cheng J.Q. J. Biol. Chem. 2002; 277: 35364-35370Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 21.Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar). Furthermore, it is evident from the most recent reports that the small GTPase Rheb is a direct target of tuberin and hamartin in both the Drosophila and mammalian systems (22.Stocker H. Radimerski T. Schindelholz B. Wittwer F. Belawat P. Daram P. Breuer S. Thomas G. Hafen E. Nat. Cell Biol. 2003; 5: 559-565Crossref PubMed Scopus (434) Google Scholar, 23.Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (716) Google Scholar, 24.Garami A. Zwartkruis F. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S. Hafen E. Bos J. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar). These studies demonstrate Rheb GTPase-activating protein activity for tuberin, and the loss of tuberin and/or hamartin lead to an increase in GTP-bound Rheb, which in turn leads to activation of the mammalian target of rapamycin/S6K/4E-binding protein signaling pathway. Thus, recent studies have placed tuberin and hamartin in growth signaling pathways; however, critical CNS functions of these proteins remain unknown. Our recent work demonstrates that unlike kidney lesions, CNS lesions such as cortical tubers do not display a second somatic mutation and other mechanisms might play a role during tumorigenesis in the CNS (25.Niida Y. Stemmer-Rachamimov A.O. Logrip M. Tapon D. Perez R. Kwiatkowski D.J. Sims K. MacCollin M. Louis D.N. Ramesh V. Am. J. Hum. Genet. 2001; 69: 493-503Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Furthermore, we have shown that the tuberin-hamartin complex exists with neurofilament light chain and ezrin, radixin, and moesin (ERM) proteins in the growth cone and could play a role in neuronal migration (26.Haddad L.A. Smith N. Bowser M. Niida Y. Murthy V. Gonzalez-Agosti C. Ramesh V. J. Biol. Chem. 2002; 277: 44180-44186Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In our efforts to understand the tuberin-hamartin complex in the CNS, we have identified Pam (protein-associated with Myc) (27.Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9172-9177Crossref PubMed Scopus (115) Google Scholar) as a physiologically relevant tuberin interactor and have shown that Pam exists in complex with tuberin-hamartin in the CNS. Our results also demonstrate that HIW, the Drosophila homolog of Pam, can genetically interact with dTsc1-dTsc2 in the fly. The evidence that the Pam homologs in worm (RPM-1) (28.Zhen M. Huang X. Bamber B. Jin Y. Neuron. 2000; 26: 331-343Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 29.Schaefer A.M. Hadwiger G.D. Nonet M.L. Neuron. 2000; 26: 345-356Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) and in Drosophila (HIW) (30.Wan H.I. DiAntonio A. Fetter R.D. Bergstrom K. Strauss R. Goodman C.S. Neuron. 2000; 26: 313-329Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar) are important regulators of synaptic growth suggests that the interaction of Pam with tuberin-hamartin will be highly relevant within the CNS. cDNA Constructs—For the yeast two-hybrid screen, human tuberin (aa 449-1000) was amplified and cloned into the two-hybrid vector pEG202 (31.Brent R. Finley Jr, R.L. Annu. Rev. Genet. 1997; 31: 663-704Crossref PubMed Scopus (118) Google Scholar). For domain mapping, tuberin deletion the Tuberin was also as a protein into Pam in vector was and cloned into in was a from Q. of was also cloned as a protein into vector The of of the was confirmed by was with the tuberin and human was to the activation domain of in the and on A. Gonzalez-Agosti C. E. D. C. F. J. Ramesh V. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). which activity within days on and within by and for with in the tuberin and hamartin and V. Stemmer-Rachamimov A.O. L.A. J.E. R.L. Smith N. Louis D.N. Ramesh V. 2001; PubMed Scopus Google University of from F. of and a human Pam (aa was in and the Cell and of and cortical from embryonic embryonic rat brain cultured as (26.Haddad L.A. Smith N. Bowser M. Niida Y. Murthy V. Gonzalez-Agosti C. Ramesh V. J. Biol. Chem. 2002; 277: 44180-44186Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For cultured with in for or and in of tuberin in and was into with with the and expressed E. and of Pam and tuberin, and embryonic rat brain in with with either or on and to with as (26.Haddad L.A. Smith N. Bowser M. Niida Y. Murthy V. Gonzalez-Agosti C. Ramesh V. J. Biol. Chem. 2002; 277: 44180-44186Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and and from by for tuberin, and with with containing and with or by (26.Haddad L.A. Smith N. Bowser M. Niida Y. Murthy V. Gonzalez-Agosti C. Ramesh V. J. Biol. Chem. 2002; 277: 44180-44186Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). with the and in For with and or was with Cells with or by and with the to a and of or with fly with and and an N. Ito N. Dickson B.J. Treisman J.E. Hariharan I.K. Cell. 2001; 105: 345-355Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). The and (30.Wan H.I. DiAntonio A. Fetter R.D. Bergstrom K. Strauss R. Goodman C.S. Neuron. 2000; 26: 313-329Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar) by and of Pam as a of proteins that associate with tuberin, we a with tuberin (aa 449-1000) a yeast two-hybrid was on a of with two the of Pam, a protein identified as a with Myc, to (27.Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9172-9177Crossref PubMed Scopus (115) Google Scholar). The of interaction was in the two-hybrid by activation of and with a of which and of which The of Pam the amino The amino of Pam are highly conserved with its homologs in (RPM-1) and Drosophila These are characterized by the of a RING zinc finger motif the motif the interaction with tuberin, we the mammalian expressed tuberin and proteins of Pam and the In to the our results that the interaction tuberin and was with was as a control We a human Pam (aa which the Pam protein expressed in the of the Pam with the the in vivo association of tuberin and Pam, we in and rat embryonic brain. was in to Pam, which cDNA has an of amino with a of (27.Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9172-9177Crossref PubMed Scopus (115) Google Scholar). of tuberin an by that Pam with tuberin in and embryonic rat brain and in rat brain a of Pam was in tuberin and hamartin together of used in these and was as a In RING was not the the of the these results that Pam with tuberin in and in vivo and may with the tuberin-hamartin complex in brain. the Pam in the for the interaction of tuberin with Pam, deletion within TSC2 and in two-hybrid The tuberin (aa 449-1000) as well as a the whereas tuberin a of of and These results suggest that the tuberin domain encoding is for the interaction the two Pam is in Pam by in cultured cortical and in the rat brain. Pam a of in cultured for days in Pam was in from which it In rat brain, Pam was on embryonic 16 with with in the of an ∼350-kDa protein with was observed in the postnatal the of which to be of Pam in the of Pam in cortical by of Pam was in the cell which is with was observed the neurites in and and was the was with the used as of Pam was confirmed in by with a neuronal that with Pam The synaptic protein a with Pam in the of cell and the neurites of Pam with the Complex in and Pam and the tuberin-hamartin complex in within cultured neurons. Tuberin is with a in the cell evidence for tuberin to the has been through its association with X. Onda H. Kwiatkowski D.J. Noonan D.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. D. N. T. 2002; PubMed Scopus Google Scholar). Tuberin also the Pam with tuberin and hamartin the neurites and in the growth It is that Pam with hamartin in one of the the of a growth cone We have that hamartin exists together with the of proteins in growth (26.Haddad L.A. Smith N. Bowser M. Niida Y. Murthy V. Gonzalez-Agosti C. Ramesh V. J. Biol. Chem. 2002; 277: 44180-44186Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). that Pam with the proteins in the growth cone Pam HIW Tsc1·Tsc2 in has been that co-expression of dTsc1 and dTsc2 genes in the the results in a of cell and cell small was shown to be by S6K and (10.Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 11.Gao X. Pan D. Genes Dev. 2001; 15: 1383-1392Crossref PubMed Scopus (390) Google Scholar, 12.Tapon N. Ito N. Dickson B.J. Treisman J.E. Hariharan I.K. Cell. 2001; 105: 345-355Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). We used to can genetically activity in vivo of in results in with of the and of which are from genetic and have genetic a genetic interaction and the The of by the of the that negatively Tsc1·Tsc2 activity in Drosophila to the control shown and In we have identified Pam as an for tuberin and have an association the tuberin-hamartin complex and Pam in the CNS. Pam was identified as a for (27.Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9172-9177Crossref PubMed Scopus (115) Google Scholar). Pam as a of in with in brain (27.Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9172-9177Crossref PubMed Scopus (115) Google Scholar). Murthy and V. In in brain high of Pam in of and of and H. K. S. D. Res. Dev. Res. 2002; PubMed Scopus Google Scholar). Pam a protein with these the most include of (aa and (aa a (aa a RZF, and two the domain (aa (27.Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9172-9177Crossref PubMed Scopus (115) Google Scholar). In a small of Pam (aa was as an interactor of and the domain of Pam was shown to be a of activity K. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we show that the Pam domain the and zinc is for its interaction with tuberin. Thus, it is evident that Pam, through could associate with a of in Our results demonstrate that Pam may have two forms in the rat CNS, an ∼450-kDa form expressed in early embryonic stages and an ∼350-kDa form observed in the postnatal period. the of the protein both The two forms may of or the studies are to the of the two In rat brain, we have observed the of tuberin and hamartin to be during with both proteins expressed in the nervous V. Stemmer-Rachamimov A.O. L.A. J.E. R.L. Smith N. Louis D.N. Ramesh V. 2001; PubMed Scopus Google Scholar). Although Pam is expressed in brain, its function in the mammalian CNS unknown. the functions of Pam homologs in Drosophila and C. are well the genetic studies in these HIW synaptic growth the in display and complex in the and HIW is to the synaptic growth (30.Wan H.I. DiAntonio A. Fetter R.D. Bergstrom K. Strauss R. Goodman C.S. Neuron. 2000; 26: 313-329Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). RPM-1, the worm homolog of Pam, is also shown to play a role during of function mutations in the presynaptic in (28.Zhen M. Huang X. Bamber B. Jin Y. Neuron. 2000; 26: 331-343Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 29.Schaefer A.M. Hadwiger G.D. Nonet M.L. Neuron. 2000; 26: 345-356Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). These results suggest that the function of these proteins as regulators of and growth are conserved fly and Our results here demonstrate that HIW can genetically interact with and negatively regulate Tsc1·Tsc2 in Drosophila. Pam, Drosophila HIW, and C. RPM-1 a of proteins that might play an role in the nervous Although the these proteins is the one of the most conserved is in the C terminus containing the zinc finger and a which we identified as the of Pam that with tuberin. the finger domain is in a of E3 ubiquitin A.M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, a genetic interaction has been identified the and that synaptic may be by a and regulators of also ubiquitin ligase activity to A. A.M. Goodman C.S. 2001; PubMed Scopus Google the that Pam may function as a ubiquitin of reports have within the that Akt phosphorylates and tuberin in a in both mammals and fly (14.Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Molecular Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar, 16.Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 20.Dan H.C. Sun M. Yang L. Feldman R.I. Sui X.M. Ou C.C. Nellist M. Yeung R.S. Halley D.J. Nicosia S.V. Pledger W.J. Cheng J.Q. J. Biol. Chem. 2002; 277: 35364-35370Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 21.Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar). TSC2 has Akt phosphorylation of which and are conserved in dTsc2 and Mutations of these to in both systems show phosphorylation (14.Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Molecular Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar, 21.Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar). phosphorylation was also shown to degradation of the tuberin-hamartin and the for a recent J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) that activation of Akt the ubiquitination and degradation of proteins containing Akt substrate tuberin. The by which Akt tuberin for is unknown. Akt the E3 ubiquitin ligase activity of RING activity is implicated in degradation of and R. Y. M. 2002; PubMed Scopus Google Scholar, L. S. C. J. 2002; PubMed Scopus Google Scholar, Y. W. Y. B. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). Studies on ubiquitination and degradation of the suggest that phosphorylation of by Akt to resulting in its ubiquitination and degradation L. S. C. J. 2002; PubMed Scopus Google Scholar). phosphorylation is to play a critical role in the of protein ubiquitination by a motif for an E3 ligase E. A. M. Nat. Cell Biol. 1999; PubMed Scopus Google Scholar, D. G. B. M. M. A. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). of tuberin by Akt is within the tuberin domain (aa that the interaction with the Pam RING finger domain. it is that Pam may function as an E3 ligase for tuberin and regulate the ubiquitination and proteasomal degradation of the tuberin-hamartin complex particularly in the CNS Although the size of Pam a for its function as an E3 ubiquitin studies will in the of the tuberin-hamartin complex is and the complex is by We Bowser for in of and of our for on the