Advertisement

Identification of Functional Domains in the Neuronal Cdk5 Activator Protein*

  • RandyY. C. Poon
    Footnotes
    Affiliations
    From the ‡ Salk Institute for Biological Studies, La Jolla, California 92037 and
    Search for articles by this author
  • John Lew
    Affiliations
    ¶Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093
    Search for articles by this author
  • Tony Hunter
    Correspondence
    American Cancer Society Research Professor. To whom correspondence should be addressed.
    Affiliations
    From the ‡ Salk Institute for Biological Studies, La Jolla, California 92037 and
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the United States Public Health Service Grants CA14195 and CA39780 (to T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Fellow of the International Human Frontier Science Program. Current address: Dept. of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
    1 The abbreviations used are: CDKcyclin-dependent kinaseCAKCDK-activating kinaseGSHglutathioneGSTglutathione S-transferasePAStaphylococcus aureus protein APAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reaction.
Open AccessPublished:February 28, 1997DOI:https://doi.org/10.1074/jbc.272.9.5703
      Cyclin-dependent kinase 5 (Cdk5) is activated by the neuronal-specific activator protein, p35. In contrast to the activation of typical CDKs by cyclin subunits, p35·;Cdk5 was not further activated by the CDK-activating kinase (CAK) and was neither phosphorylated nor inhibited by the Tyr-15-specific Wee1 kinase. The previously identified proteolytic active fragment of p35, p25 (residues 91-307) as well as the slightly smaller fragment containing residues 109-291, was found to be sufficient to bind and activate Cdk5. Other CDKs, including Cdk2, associated weakly with p25. However, their kinase activity was only activated to the low level observed for cyclin A·;Cdk2 without Thr-160 phosphorylation, and phosphorylation of Thr-160 in Cdk2 did not activate the p25·;Cdk2 complex further. We have identified distinct regions in p35 required for binding to Cdk5 or activation of Cdk5. Residues ∼150-200 of p35 were sufficient for binding to Cdk5, but residues ∼279-291 were needed in addition for activation of Cdk5 in vitro.

      INTRODUCTION

      Cyclins and cyclin-dependent kinases (CDKs)
      The abbreviations used are: CDK
      cyclin-dependent kinase
      CAK
      CDK-activating kinase
      GSH
      glutathione
      GST
      glutathione S-transferase
      PA
      Staphylococcus aureus protein A
      PAGE
      polyacrylamide gel electrophoresis
      PCR
      polymerase chain reaction.
      are key regulators of the eukaryotic cell cycle (
      • Murray A.
      • Hunt T.
      ). Cdc2 is associated with B-type cyclins and regulates M phase (
      • King R.W.
      • Jackson P.K.
      • Kirschner M.W.
      ). Cdk2 is associated with A- and E-type cyclins, and the respective complexes are believed to control the S phase and G1-S transition, respectively (
      • Sherr C.J.
      ,
      • Heichman K.A.
      • Roberts J.M.
      ). Cdk4 and Cdk6 are associated with the D-type cyclins and are important for G1 progression (
      • Sherr C.J.
      ).
      The activity of CDKs is tightly regulated by an intricate system of protein-protein interaction and phosphorylation (
      • Morgan D.O.
      ). The activation of CDKs, by definition, requires the association with cyclin partners. Full activation of CDKs requires in addition the phosphorylation of Thr-161/Thr-160, which lies in the activating T-loop in the crystal structure of Cdk2 (
      • Russo A.A.
      • Jeffrey P.D.
      • Pavletich N.P.
      ,
      • De Bondt H.L.
      • Rosenblatt J.
      • Jancarik J.
      • Jones H.D.
      • Morgan D.O.
      • Kim S.-H.
      ). The Thr-161/Thr-160 residue is phosphorylated by the CDK-activating kinase (CAK), which is composed of a cyclin H-Cdk7 complex and a RING finger protein subunit MAT1 (
      • Poon R.Y.C.
      • Hunter T.
      ). The activity of CDKs can be inhibited by phosphorylation of Thr-14 and Tyr-15 by the Wee1 and Myt1 protein kinases (
      • Mueller P.R.
      • Coleman T.R.
      • Kumagai A.
      • Dunphy W.G.
      ). Furthermore, CDKs can be inactivated by binding to CDK inhibitors like those from the p21cip1/WAF1 family (p21cip1/WAF1, p27kip1, and p57kip2) and the p16INK4A family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) (
      • Sherr C.J.
      • Roberts J.M.
      ).
      It has become clear that not all cyclins and CDKs function in cell cycle control. Examples of cyclins and CDKs that function in non-cell cycle regulating events are mounting; for instance, cyclin H·;Cdk7 and cyclin C·;Cdk8 in the TFIIH subunit of the RNA polymerase II holoenzyme have possible roles in phosphorylating the C-terminal domain of RNA polymerase II (
      • Poon R.Y.C.
      • Hunter T.
      ), and p35·;Cdk5 has a role in neurite outgrowth in postmitotic neurons (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.M.
      • Tsai L.-H.
      ).
      Cdk5 was identified as a CDK-related protein PSSALRE (
      • Meyerson M.
      • Ender H.H.
      • Wu C.-L.
      • Su L.-K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.-H.
      ) and one of the CDK partners of cyclin D in human normal diploid fibroblasts (
      • Xiong Y.
      • Zhang H.
      • Beach D.
      ). There is no other indication, however, that Cdk5 is active or functions in the normal cell cycle control (
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.
      • Chae T.
      • Harlow E.
      ). Active Cdk5 was first purified from brain extracts as a proline-directed protein kinase (
      • Lew J.
      • Beaudette K.
      • Litwin C.M.E.
      • Wang J.H.
      ). The purified protein kinase contains 33- and 25-kDa subunits; the 33-kDa subunit was later identified as the Cdc2-related kinase Cdk5 (
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ). Cdk5 was similarly identified as a neuronal protein kinase capable of phosphorylating the KSPXK sequence motif in neurofilament proteins NF-H, NF-M (
      • Shetty K.T.
      • Link W.T.
      • Pant H.C.
      ,
      • Hellmich M.R.
      • Pant H.C.
      • Wada E.
      • Battey J.F.
      ,
      • Sun D.
      • Leung C.L.
      • Liem R.K.H.
      ), and the tau protein (
      • Kobayashi S.
      • Ishiguro K.
      • Omori A.
      • Takamatsu M.
      • Arioka M.
      • Imahori K.
      • Uchida T.
      ). Phosphorylation of tau by Cdk5 is particularly interesting because abnormally phosphorylated tau is the major component of the paired helical filaments, which accumulate in the brains of Alzheimer patients (
      • Mandelkow E.-M.
      • Mandelkow E.
      ). Cdk5 is able to phosphorylate tau on sites that are abnormally phosphorylated in Alzheimer's paired helical filaments (
      • Baumann K.
      • Mandelkow E.M.
      • Biernat J.
      • Piwnica-Worms H.
      • Mandelkow E.
      ,
      • Paudel H.K.
      • Lew J.
      • Ali Z.
      • Wang J.H.
      ). The 25-kDa subunit of the Cdk5 kinase (
      • Lew J.
      • Beaudette K.
      • Litwin C.M.E.
      • Wang J.H.
      ,
      • Ishiguro K.
      • Kobayashi S.
      • Omori A.
      • Takamatsu M.
      • Yonekura S.
      • Anzai K.
      • Imahori K.
      • Uchida T.
      ) was later found to be a proteolytic fragment of a larger 35-kDa protein (p35) (
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.
      • Chae T.
      • Harlow E.
      ,
      • Uchida T.
      • Ishiguro K.
      • Ohnuma J.
      • Takamatsu M.
      • Yonekura S.
      • Imahori K.
      ,
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ). An isoform of p35 that shares 57% amino acid identity to p35 has been identified, and its mRNA is predominantly expressed in the hippocampus (
      • Tang D.
      • Yeung J.
      • Lee K.-Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ).
      The complex p35·;Cdk5 is only active as a histone H1 kinase in postmitotic neurons (
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.
      • Chae T.
      • Harlow E.
      ,
      • Tsai L.H.
      • Takahashi T.
      • Caviness Jr., V.S.
      • Harlow E.
      ). Moreover, p35·;Cdk5 appears to be important for normal neuronal function; its kinase activity increases during neurogenesis, and it is essential for neurite outgrowth during neuronal differentiation (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.M.
      • Tsai L.-H.
      ). A fundamental role for p35·;Cdk5 is supported by the findings that targeted disruption of the p35 gene in the mouse leads to severe defects in laminar organization of neurons in the neocortex and cerebellum,
      L.-H. Tsai, personal communication.
      and targeted disruption of Cdk5 gene leads to abnormal corticogenesis, lesions in the central nervous system, and perinatal death (
      • Ohshima T.
      • Ward J.M.
      • Huh C.-G.
      • Longenecker G.
      • Veeranna Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ). Although p35 shares little sequence similarity to cyclin, computer modeling predicts that p35 may fold into cyclin-like tertiary structure and activate Cdk5 in a manner similar to a cyclin (
      • Brown N.R.
      • Noble M.E.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ). This is reminiscent of the two repeating cyclin folds found in cyclin A, which lack sequence homology but nevertheless have an identical structure (
      • Jeffrey P.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massagué J.
      • Pavletich N.P.
      ). Even though p35 may adopt a cyclin-like structure and can activate Cdk5, the regulation of p35·;Cdk5 appears to be very much distinct from other cyclin·;CDK complexes. Activation of typical cyclin·;CDK complexes requires phosphorylation of Thr-161 or equivalent residues, but p35·;Cdk5 is active as a histone H1 kinase in the absence of phosphorylation by other protein kinases (
      • Qi Z.
      • Huang Q.-Q.
      • Lee K.-Y.
      • Lew J.
      • Wang J.H.
      ), despite the fact that the surrounding sequences of Ser-159 in Cdk5 (the Thr-161-equivalent residue) are similar to other CDKs. Moreover, no autophosphorylation of Cdk5 was observed under those conditions (
      • Qi Z.
      • Huang Q.-Q.
      • Lee K.-Y.
      • Lew J.
      • Wang J.H.
      ). Unlike most other cyclin·;CDK complexes, p35·;Cdk5 is not inhibited by the p21cip1/WAF1 (
      • Harper J.W.
      • Elledge S.J.
      • Keyomarsi K.
      • Dynlacht B.
      • Tsai L.-H.
      • Zhang P.
      • Dobrowolski S.
      • Bai C.
      • Connell-Crowley L.
      • Swindell E.
      • Fox M.P.
      • Wei N.
      ) or p27kip1 (
      • Lee M.H.
      • Nikolic M.
      • Baptista C.A.
      • Lai E.
      • Tsai L.H.
      • Massagué J.
      ) CDK inhibitors. There is some evidence, however, that a population of p35·;Cdk5 exists as an inactive form within a macromolecular structure, suggesting p35·;Cdk5 may bind to inhibitors in the brain (
      • Lee K.-Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      ).
      Here we investigated the mechanism of activation of Cdk5 in vitro. We have defined distinct regions in p35 required either for binding to Cdk5 or activation of Cdk5. Residues ∼150-200 of p35 were sufficient for binding to Cdk5 in vitro, but residues ∼279-291 were in addition critical for activation of Cdk5. The regulation of p35·;Cdk5 is likely to be very different from that of other cyclin·;CDK pairs, because unlike cyclin A·;Cdk2, p35·;Cdk5 was not activated by CAK or inhibited by Wee1 under the same conditions.

      DISCUSSION

      Judging by sequence homology alone, Cdk5 is a typical CDK-like protein kinase with nothing in particular to indicate a major difference from the rest of the CDK family (
      • Meyerson M.
      • Ender H.H.
      • Wu C.-L.
      • Su L.-K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.-H.
      ). Indeed, as with Cdk4 and Cdk6, Cdk5 has been found to associate with D-type cyclins in normal diploid fibroblasts (
      • Xiong Y.
      • Zhang H.
      • Beach D.
      ). But as yet, there is no indication that cyclin D·;Cdk5 functions in the normal cell cycle. Unlike dominant-negative mutants of Cdk2 and Cdc2, overexpression of a dominant-negative Cdk5 mutant does not cause cell cycle arrest (
      • van den Heuvel S.
      • Harlow E.
      ), and no histone H1 kinase activity associated with Cdk5 can be detected except in post-mitotic neuronal cells.
      What then is the molecular basis of the uniqueness of Cdk5 compared with other CDKs? If Cdk5 function is important for neuronal differentiation, as indicated by recent evidence (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.M.
      • Tsai L.-H.
      ,
      • Ohshima T.
      • Ward J.M.
      • Huh C.-G.
      • Longenecker G.
      • Veeranna Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ), then Cdk5 must be regulated by very different mechanisms compared with other cell cycle-regulating cyclin·;CDK complexes. Cell cycle-regulating cyclins and CDKs are generally turned off during neuronal differentiation (
      • Buchkovich K.J.
      • Ziff E.B.
      ,
      • Kranenburg O.
      • Scharnhorst V.
      • Van der Eb A.J.
      • Zantema A.
      ), since cell cycle arrest appears to be a prerequisite for differentiation. The activity of cell cycle regulatory cyclin·;CDKs can be down-regulated by several mechanisms, including Thr-14/Tyr-15 phosphorylation by protein kinases such as Wee1, decreased Thr-160 phosphorylation, and binding of CDK inhibitors like p21cip1/WAF1 and p27kip1. Cdk5 is immune to these inhibitory mechanisms because it is bound to an atypical partner p35/p25. The levels of p21cip1/WAF1 and p27kip1 are elevated during differentiation in tissues such as neurons and muscles and may play a role in turning off cyclin·;CDK activities. (
      • Skapek S.X.
      • Rhee J.
      • Spicer D.B.
      • Lassar A.B.
      ,
      • Guo K.
      • Wang J.
      • Andrés V.
      • Smith R.C.
      • Walsh K.
      ). Consistent with a requirement for down-regulation of CDK activity in neuronal cell differentiation, expression of Cdk2 blocks nerve growth factor-induced differentiation of PC12 cells (
      • Dobashi Y.
      • Kudoh T.
      • Matsumine A.
      • Toyoshima K.
      • Akiyama T.
      ). During nerve growth factor-induced differentiation of PC12 cells, cyclin D1 expression is increased but cyclin D1-associated kinase activity is not induced, possibly due to inhibition by p21cip1/WAF1 or related proteins (
      • Dobashi Y.
      • Kudoh T.
      • Matsumine A.
      • Toyoshima K.
      • Akiyama T.
      ,
      • Yan G.Z.
      • Ziff E.B.
      ,
      • van Grunsven L.A.
      • Thomas A.
      • Urdiales J.L.
      • Machenaud S.
      • Choler P.
      • Durand I.
      • Rudkin B.B.
      ). Moreover, overexpression of p27kip1 alone is sufficient to induce neuronal differentiation in a mouse neuroblastoma cell line (
      • Kranenburg O.
      • Scharnhorst V.
      • Van der Eb A.J.
      • Zantema A.
      ). The kinase activity of p35·;Cdk5, however, is not inhibited by p21cip1/WAF1 or p27kip1 (
      • Harper J.W.
      • Elledge S.J.
      • Keyomarsi K.
      • Dynlacht B.
      • Tsai L.-H.
      • Zhang P.
      • Dobrowolski S.
      • Bai C.
      • Connell-Crowley L.
      • Swindell E.
      • Fox M.P.
      • Wei N.
      ,
      • Lee M.H.
      • Nikolic M.
      • Baptista C.A.
      • Lai E.
      • Tsai L.H.
      • Massagué J.
      ). We also found that the activity of bacterially expressed p25·;Cdk5 is not inhibited by recombinant p21cip1/WAF1 or p27kip1.
      R. Y. C. Poon, unpublished observations.
      Not only is p25·;Cdk5 not regulated by p21cip1/WAF1/p27kip1 family, but as we show here p25·;Cdk5 kinase activity is not dependent on CAK nor is it negatively regulated by Wee1. It is of course possible that neurons contain specific versions of CAK, Wee1, or inhibitors that are capable of regulating p25·;Cdk5. This is particularly relevant because it has been observed that a population of p35·;Cdk5 is not active in brain extracts (
      • Lee K.-Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      ). Moreover, an activity capable of phosphorylating Thr-14 in Cdc2 has been purified from thymus that can also phosphorylate and inhibit p25·;Cdk5 (
      • Matsuura I.
      • Wang J.H.
      ).
      Here we show that a deletion of the C terminus of p25 (beyond residue 291) abolished its ability to activate Cdk5, despite the fact that it could still associate with Cdk5. The C-terminal deletion may either disrupt the conformation of the whole activating domain without affecting the binding domain, or the residues at the C terminus are directly required for Cdk5 activation. However, residues 279-291 in p35 do not show any similarity to any other known protein including the cyclin family. Interestingly, the other isoform of p35, p39, is very similar in sequence to p35 up to amino acid residue 291, after which there is a 24-amino acid insertion in p39 (
      • Tang D.
      • Yeung J.
      • Lee K.-Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ). The ability of some p35 mutants described here to bind but not activate Cdk5 is in marked contrast to the situation with cyclin A, where all reported mutants that bind Cdc2 and Cdk2 also activate their kinase activities (
      • Kobayashi H.
      • Steward E.
      • Poon R.
      • Adamczewski J.P.
      • Gannon J.
      • Hunt T.
      ,
      • Lees E.M.
      • Harlow E.
      ), suggesting a fundamental difference in the activation of Cdc2/Cdk2 by cyclin A and Cdk5 by p35. Indeed, the p25 truncation mutants characterized here are the first dominant-negative mutants of a CDK activating subunit to be described. The CΔ251 p35 mutant could prove useful in conjunction with Cdk5 mutants in probing p35·;Cdk5 function in vivo.
      Another group
      D. Tang, A. C. S. Chun, M. Zhang, and J. H. Wang, submitted for publication.
      have recently obtained results similar to ours and shown that a small deletion of the C-terminal region of p25 is sufficient to prevent Cdk5 activation. However, in contrast to our results, they found that a minimal deletion of the C-terminal region of p25 also abolished Cdk5 binding. This difference may due to the fact that we used a slightly longer version of p25 at the N terminus (from residue 109) than they did (from residue 145) or that the 12 vector-derived residues at the end of our fusion proteins have some effect; it is also possible that it is due to the more stringent binding assay conditions used in their studies.
      Despite the fact that there is very little sequence similarity between p35 and cyclins, it has been predicted that p35 may have a cyclin-like structure (
      • Brown N.R.
      • Noble M.E.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ). The cyclin box region of cyclin A has an α-helical fold comprised of five α-helices, and this fold is repeated in the C-terminal region following the cyclin box but shares little sequence similarity with the N-terminal fold (
      • Brown N.R.
      • Noble M.E.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ,
      • Jeffrey P.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massagué J.
      • Pavletich N.P.
      ). The region in p35 that is predicted to have a cyclin-fold structure is between residues ∼135-227, which covers the region we found to be important in binding to Cdk5 (∼150-200), although the minimal Cdk5 binding domain would lack the predicted helices 1 and 5. The C-terminal Cdk5-activating domain would be predicted to lie outside the “cyclin box” region and may have an interesting structure that distinguishes p35 from other cyclins. The fact that p35·;Cdk5 activity does not require phosphorylation of Ser-159 in the Cdk5 activation loop implies that p35 interacts with this loop more extensively than the cyclins do, acting instead of phosphorylation to hold the activation loop in an active conformation. The regions in p35 that diverge in structure from other cyclins are presumably responsible for the different mechanism of Cdk5 regulation. The lack of similarity between p35 and cyclins may well account for the lack of binding to and inhibition by p21cip1/WAF1 and p27kip1, since a key element of the inhibitory mechanism requires binding of the inhibitor protein to a groove in the cyclin (
      • Russo A.A.
      • Jeffrey P.D.
      • Patten A.K.
      • Massagué J.
      • Pavletich N.P.
      ). A specialized mechanism of p35 association with the Cdk5 activation loop could explain the lack of Cdk5 phosphorylation by CAK and Wee1.

      Acknowledgments

      We thank D. Tang, A. C. S. Chun, M. Zhang, and J. H. Wang for communication of unpublished data.

      REFERENCES

        • Murray A.
        • Hunt T.
        The Cell Cycle. Oxford University Press, Oxford1993
        • King R.W.
        • Jackson P.K.
        • Kirschner M.W.
        Cell. 1994; 79: 563-571
        • Sherr C.J.
        Cell. 1994; 79: 551-555
        • Heichman K.A.
        • Roberts J.M.
        Cell. 1994; 79: 557-562
        • Morgan D.O.
        Nature. 1995; 374: 131-134
        • Russo A.A.
        • Jeffrey P.D.
        • Pavletich N.P.
        Nat. Struct. Biol. 1996; 3: 696-700
        • De Bondt H.L.
        • Rosenblatt J.
        • Jancarik J.
        • Jones H.D.
        • Morgan D.O.
        • Kim S.-H.
        Nature. 1993; 363: 595-602
        • Poon R.Y.C.
        • Hunter T.
        Curr. Biol. 1995; 5: 1243-1247
        • Mueller P.R.
        • Coleman T.R.
        • Kumagai A.
        • Dunphy W.G.
        Science. 1995; 270: 86-90
        • Sherr C.J.
        • Roberts J.M.
        Genes Dev. 1995; 9: 1149-1163
        • Nikolic M.
        • Dudek H.
        • Kwon Y.T.
        • Ramos Y.F.M.
        • Tsai L.-H.
        Genes Dev. 1996; 10: 816-825
        • Meyerson M.
        • Ender H.H.
        • Wu C.-L.
        • Su L.-K.
        • Gorka C.
        • Nelson C.
        • Harlow E.
        • Tsai L.-H.
        EMBO J. 1992; 11: 2909-2917
        • Xiong Y.
        • Zhang H.
        • Beach D.
        Cell. 1992; 71: 504-514
        • Tsai L.H.
        • Delalle I.
        • Caviness V.S.
        • Chae T.
        • Harlow E.
        Nature. 1994; 371: 419-423
        • Lew J.
        • Beaudette K.
        • Litwin C.M.E.
        • Wang J.H.
        J. Biol. Chem. 1992; 267: 13383-13390
        • Lew J.
        • Winkfein R.J.
        • Paudel H.K.
        • Wang J.H.
        J. Biol. Chem. 1992; 267: 25922-25926
        • Shetty K.T.
        • Link W.T.
        • Pant H.C.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6844-6848
        • Hellmich M.R.
        • Pant H.C.
        • Wada E.
        • Battey J.F.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10867-10871
        • Sun D.
        • Leung C.L.
        • Liem R.K.H.
        J. Biol. Chem. 1996; 271: 14245-14251
        • Kobayashi S.
        • Ishiguro K.
        • Omori A.
        • Takamatsu M.
        • Arioka M.
        • Imahori K.
        • Uchida T.
        FEBS Lett. 1993; 335: 171-175
        • Mandelkow E.-M.
        • Mandelkow E.
        Trends Biochem. Sci. 1993; 18: 480-483
        • Baumann K.
        • Mandelkow E.M.
        • Biernat J.
        • Piwnica-Worms H.
        • Mandelkow E.
        FEBS Lett. 1993; 336: 417-424
        • Paudel H.K.
        • Lew J.
        • Ali Z.
        • Wang J.H.
        J. Biol. Chem. 1993; 268: 23512-23518
        • Ishiguro K.
        • Kobayashi S.
        • Omori A.
        • Takamatsu M.
        • Yonekura S.
        • Anzai K.
        • Imahori K.
        • Uchida T.
        FEBS Lett. 1994; 342: 203-208
        • Uchida T.
        • Ishiguro K.
        • Ohnuma J.
        • Takamatsu M.
        • Yonekura S.
        • Imahori K.
        FEBS Lett. 1994; 355: 35-40
        • Lew J.
        • Huang Q.Q.
        • Qi Z.
        • Winkfein R.J.
        • Aebersold R.
        • Hunt T.
        • Wang J.H.
        Nature. 1994; 371: 423-426
        • Tang D.
        • Yeung J.
        • Lee K.-Y.
        • Matsushita M.
        • Matsui H.
        • Tomizawa K.
        • Hatase O.
        • Wang J.H.
        J. Biol. Chem. 1995; 270: 26897-26903
        • Tsai L.H.
        • Takahashi T.
        • Caviness Jr., V.S.
        • Harlow E.
        Development. 1993; 119: 1029-1040
        • Ohshima T.
        • Ward J.M.
        • Huh C.-G.
        • Longenecker G.
        • Veeranna Pant H.C.
        • Brady R.O.
        • Martin L.J.
        • Kulkarni A.B.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178
        • Brown N.R.
        • Noble M.E.
        • Endicott J.A.
        • Garman E.F.
        • Wakatsuki S.
        • Mitchell E.
        • Rasmussen B.
        • Hunt T.
        • Johnson L.N.
        Structure. 1995; 3: 1235-1247
        • Jeffrey P.
        • Russo A.A.
        • Polyak K.
        • Gibbs E.
        • Hurwitz J.
        • Massagué J.
        • Pavletich N.P.
        Nature. 1995; 376: 313-320
        • Qi Z.
        • Huang Q.-Q.
        • Lee K.-Y.
        • Lew J.
        • Wang J.H.
        J. Biol. Chem. 1995; 270: 10847-10854
        • Harper J.W.
        • Elledge S.J.
        • Keyomarsi K.
        • Dynlacht B.
        • Tsai L.-H.
        • Zhang P.
        • Dobrowolski S.
        • Bai C.
        • Connell-Crowley L.
        • Swindell E.
        • Fox M.P.
        • Wei N.
        Mol. Biol. Cell. 1995; 6: 387-400
        • Lee M.H.
        • Nikolic M.
        • Baptista C.A.
        • Lai E.
        • Tsai L.H.
        • Massagué J.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3259-3263
        • Lee K.-Y.
        • Rosales J.L.
        • Tang D.
        • Wang J.H.
        J. Biol. Chem. 1996; 271: 1538-1543
        • Poon R.Y.C.
        • Yamashita K.
        • Adamczewski J.P.
        • Hunt T.
        • Shuttleworth J.
        EMBO J. 1993; 12: 3123-3132
        • Poon R.Y.C.
        • Hunter T.
        Science. 1995; 270: 90-93
        • Kobayashi H.
        • Steward E.
        • Poon R.
        • Adamczewski J.P.
        • Gannon J.
        • Hunt T.
        Mol. Biol. Cell. 1992; 3: 1279-1294
        • Kobayashi H.
        • Minshull J.
        • Ford C.
        • Golsteyn R.
        • Poon R.
        • Hunt T.
        J. Cell Biol. 1991; 114: 755-765
        • Horton R.M.
        • Pease L.R.
        McPherson M.J. Directed Mutagenesis. IRL Press at Oxford University Press, Oxford1991: 217-247
        • Poon R.Y.C.
        • Toyoshima H.
        • Hunter T.
        Mol. Biol. Cell. 1995; 6: 1197-1213
        • Nielsen D.A.
        • Shapiro D.J.
        Nucleic Acids Res. 1986; 14: 5936
        • Jackson R.J.
        • Hunt T.
        Methods Enzymol. 1983; 96: 50-73
        • Poon R.Y.C.
        • Yamashita K.
        • Howell M.
        • Ershler M.A.
        • Belyavsky A.
        • Hunt T.
        J. Cell Sci. 1994; 107: 2789-2799
        • Poon R.Y.C.
        • Jiang W.
        • Toyoshima H.
        • Hunter T.
        J. Biol. Chem. 1996; 271: 13283-13291
        • Connell-Crowley L.
        • Solomon M.J.
        • Wei N.
        • Harper J.W.
        Mol. Biol. Cell. 1993; 4: 79-92
        • Gabrielli B.G.
        • Lee M.S.
        • Walker D.H.
        • Piwnica-Worms H.
        • Maller J.L.
        J. Biol. Chem. 1992; 267: 18040-18046
        • Sebastian B.
        • Kakizuka A.
        • Hunter T.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3521-3524
        • Terada Y.
        • Tatsuka M.
        • Jinno S.
        • Okayama H.
        Nature. 1995; 376: 358-362
        • van den Heuvel S.
        • Harlow E.
        Science. 1993; 262: 2050-2054
        • Buchkovich K.J.
        • Ziff E.B.
        Mol. Biol. Cell. 1994; 5: 1225-1241
        • Kranenburg O.
        • Scharnhorst V.
        • Van der Eb A.J.
        • Zantema A.
        J. Cell Biol. 1995; 131: 227-234
        • Skapek S.X.
        • Rhee J.
        • Spicer D.B.
        • Lassar A.B.
        Science. 1995; 267: 1022-1024
        • Guo K.
        • Wang J.
        • Andrés V.
        • Smith R.C.
        • Walsh K.
        Mol. Cell. Biol. 1995; 15: 3823-3828
        • Dobashi Y.
        • Kudoh T.
        • Matsumine A.
        • Toyoshima K.
        • Akiyama T.
        J. Biol. Chem. 1995; 270: 23031-23037
        • Yan G.Z.
        • Ziff E.B.
        J. Neurosci. 1995; 15: 6200-6212
        • van Grunsven L.A.
        • Thomas A.
        • Urdiales J.L.
        • Machenaud S.
        • Choler P.
        • Durand I.
        • Rudkin B.B.
        Oncogene. 1996; 12: 855-862
        • Matsuura I.
        • Wang J.H.
        J. Biol. Chem. 1996; 271: 5443-5450
        • Lees E.M.
        • Harlow E.
        Mol. Cell. Biol. 1993; 13: 1194-1201
        • Russo A.A.
        • Jeffrey P.D.
        • Patten A.K.
        • Massagué J.
        • Pavletich N.P.
        Nature. 1996; 382: 325-331