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Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination*

Open AccessPublished:December 02, 2015DOI:https://doi.org/10.1074/jbc.M115.692871
      Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death.

      Introduction

      Cyclin-dependent kinases (Cdks)
      The abbreviations used are: Cdk5, cyclin-dependent kinase 5; CHX, cycloheximide; 23R, 23 Lys residues to Arg; N7, the 7 N-terminal amino acid.
      are a family of Ser/Thr kinases that are activated by binding a regulatory subunit called cyclin. Most members of Cdks are expressed in proliferating cells to promote cell cycle progression (
      • Malumbres M.
      Cyclin-dependent kinases.
      ). In contrast, Cdk5 is activated by p35 or p39 non-cyclin proteins, which are mainly expressed in post-mitotic neurons (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ). Cdk5 is a versatile kinase that is involved in many neuronal activities, including neuronal cell layer formation, synaptic transmission, membrane trafficking, and neuron cell death (
      • Shah K.
      • Lahiri D.K.
      Cdk5 activity in the brain: multiple paths of regulation.
      ). p35 and p39 appear to share common and/or distinct functions for Cdk5, with p35 being the predominant activator. This is shown by the phenotypes of knock-out (KO) mice; p35 KO mice display abnormal neural layers in the cerebral cortex (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality.
      ), and p39 KO mice do not show apparent abnormalities, whereas p35 and p39 double KO mice are perinatal lethal with abnormal neural layers, as are the Cdk5 KO mice (
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology, and perinatal death.
      ,
      • Gilmore E.C.
      • Ohshima T.
      • Goffinet A.M.
      • Kulkarni A.B.
      • Herrup K.
      Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex.
      • Ko J.
      • Humbert S.
      • Bronson R.T.
      • Takahashi S.
      • Kulkarni A.B.
      • Li E.
      • Tsai L.H.
      p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.
      ). To understand the precise function of Cdk5-p35 in various neuronal activities, it is important to reveal the regulation mechanism of Cdk5 activity.
      As well as being cell cycle Cdks, Cdk5 is a stable protein and is expressed more abundantly than p35 in neurons (
      • Lee K.Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      Interaction of cyclin-dependent kinase 5 (Cdk5) and neuronal Cdk5 activator in bovine brain.
      ,
      • Zhu Y.S.
      • Saito T.
      • Asada A.
      • Maekawa S.
      • Hisanaga S.
      Activation of latent cyclin-dependent kinase 5 (Cdk5)-p35 complexes by membrane dissociation.
      ). Therefore, Cdk5 activity is determined primarily by the available amount of activator protein p35, and the protein amounts of p35 are regulated by the balance between synthesis and degradation (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ). Although the synthesis of p35 is stimulated by NGF or BDNF (
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1.
      ,
      • Bogush A.
      • Pedrini S.
      • Pelta-Heller J.
      • Chan T.
      • Yang Q.
      • Mao Z.
      • Sluzas E.
      • Gieringer T.
      • Ehrlich M.E.
      AKT and CDK5/p35 mediate brain derived neurotrophic factor induction of DRPP-32 in medium size spiny neuron in vitro.
      ), the degradation of p35 is carried out by proteasomes (
      • Patrick G.N.
      • Zhou P.
      • Kwon Y.T.
      • Howley P.M.
      • Tsai L.H.
      p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5), is degraded by the ubiquitin-proteasome pathway.
      ,
      • Saito T.
      • Ishiguro K.
      • Onuki R.
      • Nagai Y.
      • Kishimoto T.
      • Hisanaga S.
      Okadaic acid-stimulated degradation of p35, an activator of Cdk5, by proteasome in cultured neurons.
      ). The degradation is a major determinant of the p35 level, which is reduced by treating neurons with excitatory neurotransmitter glutamate (
      • Wei F.Y.
      • Tomizawa K.
      • Ohshima T.
      • Asada A.
      • Saito T.
      • Nguyen C.
      • Bibb J.A.
      • Ishiguro K.
      • Kulkarni A.B.
      • Pant H.C.
      • Mikoshiba K.
      • Matsui H.
      • Hisanaga S.
      Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability.
      ). p35 associates with membranes via myristoylation at the N-terminal glycine (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Asada A.
      • Yamamoto N.
      • Gohda M.
      • Saito T.
      • Hayashi N.
      • Hisanaga S.
      Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cyclin-dependent kinase 5 complexes.
      ), and this association enhances the degradation of p35 (
      • Minegishi S.
      • Asada A.
      • Miyauchi S.
      • Fuchigami T.
      • Saito T.
      • Hisanaga S.
      Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.
      ). On the other hand, p35 is cleaved by a calcium-dependent protease calpain to produce the C-terminal stable fragment p25 (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ). Although the physiological function of Cdk5-p25 has been recently reported (
      • Engmann O.
      • Hortobágyi T.
      • Thompson A.J.
      • Guadagno J.
      • Troakes C.
      • Soriano S.
      • Al-Sarraj S.
      • Kim Y.
      • Giese K.P.
      Cyclin-dependent kinase 5 activator p25 is generated during memory formation and is reduced at an early stage in Altzheiner's disease.
      ,
      • Rei D.
      • Mason X.
      • Seo J.
      • Gräff J.
      • Rudenko A.
      • Wang J.
      • Rueda R.
      • Siegert S.
      • Cho S.
      • Canter R.G.
      • Mungenast A.E.
      • Deisseroth K.
      • Tsai L.H.
      Basolateral amygdala bidirectionally modulates stress-induced hippocampal learning and memory deficits through a p25/Cdk5-dependent pathway.
      ), its abundance induces neuronal cell death in neurodegenerative diseases (
      • Cruz J.C.
      • Tsai L.H.
      Cdk5 deregulation in pathogenesis of Alzheimer disease.
      ). Rapid turnover of p35 is suggested to be a mechanism to prevent p25 production (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ). Therefore, it is particularly important to determine the degradation mechanism of p35. Interestingly, the addition of the N-terminal hepta-peptide containing the myristoylation site of p35 facilitates p35 lability (
      • Minegishi S.
      • Asada A.
      • Miyauchi S.
      • Fuchigami T.
      • Saito T.
      • Hisanaga S.
      Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.
      ), indicating that the degradation of p35 occurs selectively on membranes. Although p35 has previously been demonstrated to be post-translationally modified by ubiquitination (
      • Patrick G.N.
      • Zhou P.
      • Kwon Y.T.
      • Howley P.M.
      • Tsai L.H.
      p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5), is degraded by the ubiquitin-proteasome pathway.
      ), the E3 ligase responsible has not been identified yet in the neuron, and its degradation pathway is not completely understood.
      The ubiquitin-proteasome system is a major component of the proteolytic machinery that performs the degradation of proteins in cells (
      • Hershko A.
      • Ciechanover A.
      The ubiquitin system for protein degradation.
      ,
      • Ravid T.
      • Hochstrasser M.
      Diversity of degradation signals in the ubiquitin-proteasome system.
      ). Ubiquitin is a small protein that is tagged to substrate proteins to be degraded. The proteasome is a large complex of multicatalytic proteases that degrades proteins to small peptides. The 26S proteasome is a complex of 20S proteasome and 19S particles. The 20S proteasome is the core of the proteasome, and 19S is a regulatory particle (PA700) that recognizes and unfolds ubiquitinated proteins. The unfolded proteins are proteolyzed by being inserted into the 20S chamber. Ubiquitination is a critical step in the ubiquitin-proteasome system for selective degradation, which is catalyzed by E3 ubiquitin ligases. There are large numbers of E3 ligases with a specific substrate (
      • Tanaka K.
      • Suzuki T.
      • Hattori N.
      • Mizuno Y.
      Ubiquitin, proteasome and parkin.
      ). The E3 ligase for p35 has not been found in the brain, although in pancreatic β-cells, Pja2 has been recently reported to have E3 ligase activity to p35 (
      • Sakamaki J.
      • Fu A.
      • Reeks C.
      • Baird S.
      • Depatie C.
      • Al Azzabi M.
      • Bardeesy N.
      • Gingras A.C.
      • Yee S.P.
      • Screaton R.A.
      Role of the SIK-p35 PJA2 complex in pancreatic β-cell functional compensation.
      ).
      However, polyubiquitination is not an absolute requirement for proteasomal degradation. Ornithine decarboxylase is a well known example of ubiquitin-independent proteasomal degradation (
      • Murakami Y.
      • Matsufuji S.
      • Hayashi S.
      • Tanahashi N.
      • Tanaka K.
      Degradation of ornithine decarboxylase by the 26S proteasome.
      ,
      • Hoyt M.A.
      • Zhang M.
      • Coffino P.
      Ubiquitin-independent mechanisms of mouse ornithine decarboxylase are conserved between mammalian and fugal cells.
      ). The number of proteins susceptible to ubiquitin-independent proteasomal degradation had recently been increasing. They include thymidylate synthase, Rpn4, p21 Cdk inhibitor, p53 tumor suppressor, c-Fos, Nkx3.1, and so on (
      • Peña M.M.
      • Xing Y.Y.
      • Koli S.
      • Berger F.G.
      Role of N-terminal residues in the ubiquitin-independent degradation of human thymidylate synthase.
      • Peña M.M.
      • Melo S.P.
      • Xing Y.Y.
      • White K.
      • Barbour K.W.
      • Berger F.G.
      The intrinsically disordered N-terminal domain of thymidylate synthase targets the enzyme to the ubiquitin-independent proteasomal degradation pathway.
      ,
      • Ha S.W.
      • Ju D.
      • Xie Y.
      The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits.
      ,
      • Chen X.
      • Chi Y.
      • Bloecher A.
      • Aebersold R.
      • Clurman B.E.
      • Roberts J.M.
      N-Acetylation and ubiquitin-independent proteasomal degradation of p21 (Cip 1).
      ,
      • Tsvetkov P.
      • Reuven N.
      • Shaul Y.
      Ubiquitin-independent p53 proteasomal degradation.
      ,
      • Basbous J.
      • Jariel-Encontre I.
      • Gomard T.
      • Bossis G.
      • Piechaczyk M.
      Ubiquitin-independent versus ubiquitin-dependent proteasomal degradation of the c-Fos and Fra-1 trunscription factors: is there a unique answer?.
      • Rao V.
      • Guan B.
      • Mutton L.N.
      • Bieberich C.J.
      Proline-mediate proteasomal degradation of the prostate-specific tumor suppressor NKX3.1.
      ). Degron sequences recognized by proteasomes have been investigated in these proteins, but their degradation mechanisms and physiological meanings are largely unknown. Herein, we intended to identify the ubiquitination sites on p35 for a better understanding of its ubiquitin-proteasome-dependent degradation mechanism. Unexpectedly, however, we found that non-ubiquitinated p35 was degraded at a comparable rate to wild-type p35. Our results indicate that p35 is degraded by proteasomes by two pathways: ubiquitin-dependent and ubiquitin-independent.

      Discussion

      p35 is an unstable protein with a half-life of 30–60 min, and it is degraded by proteasomes after ubiquitination (
      • Patrick G.N.
      • Zhou P.
      • Kwon Y.T.
      • Howley P.M.
      • Tsai L.H.
      p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5), is degraded by the ubiquitin-proteasome pathway.
      ,
      • Saito T.
      • Ishiguro K.
      • Onuki R.
      • Nagai Y.
      • Kishimoto T.
      • Hisanaga S.
      Okadaic acid-stimulated degradation of p35, an activator of Cdk5, by proteasome in cultured neurons.
      ,
      • Minegishi S.
      • Asada A.
      • Miyauchi S.
      • Fuchigami T.
      • Saito T.
      • Hisanaga S.
      Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.
      ). Because the p35 protein amount is a critical determinant of Cdk5 activity, elucidating the p35 degradation mechanism is central for understanding Cdk5 functions. To this end we searched for the polyubiquitination site(s) in p35, but unexpectedly, we found that the degradation of p35 can occur without ubiquitination. We also showed that ubiquitin-independent degradation was mediated by an α-helical degron-like sequence in the C-terminal region of p35. Thus, p35 is subjected to two different degradation mechanisms: ubiquitin-dependent and ubiquitin-independent.
      In contrast to cell cycle Cdks, whose activation is regulated by its phosphorylation/dephosphorylation upon cyclin binding (
      • Morgan D.O.
      Cyclin-dependent kinases: engines, clocks, and microprocessors.
      ), Cdk5 is activated only by binding to its activation subunit p35 (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ). On the other hand, similar to cell cycle Cdks, whose inactivation is induced by the degradation of cyclin, Cdk5 is inactivated by the degradation of p35 by proteasomes. Because Cdk5 is expressed more than p35 in neurons (
      • Lee K.Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      Interaction of cyclin-dependent kinase 5 (Cdk5) and neuronal Cdk5 activator in bovine brain.
      ,
      • Zhu Y.S.
      • Saito T.
      • Asada A.
      • Maekawa S.
      • Hisanaga S.
      Activation of latent cyclin-dependent kinase 5 (Cdk5)-p35 complexes by membrane dissociation.
      ), the protein level of p35 is a limiting factor determining the total Cdk5 activity. Cyclins are typical well studied proteins to be targeted by proteasomes via ubiquitination in a cell cycle-dependent manner (
      • Benanti J.A.
      Codination of cell growth and divition by the ubiquitin-proteasome system.
      ). Therefore, it is natural to expect that p35 is also targeted by proteasomes when it is ubiquitinated. In fact, ubiquitination of p35 has been demonstrated by several previous studies by groups including ours (
      • Patrick G.N.
      • Zhou P.
      • Kwon Y.T.
      • Howley P.M.
      • Tsai L.H.
      p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5), is degraded by the ubiquitin-proteasome pathway.
      ,
      • Minegishi S.
      • Asada A.
      • Miyauchi S.
      • Fuchigami T.
      • Saito T.
      • Hisanaga S.
      Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.
      ,
      • Endo R.
      • Saito T.
      • Asada A.
      • Kawahara H.
      • Ohshima T.
      • Hisanaga S.
      Commitment of 1-methyl-4-phenylpyrinidinium ion-induced neuronal cell death by proteasome-mediated degradation of p35 cyclin-dependent kinase 5 activator.
      ). Therefore, it was surprising for us to find that the lysine-less mutant of p35 underwent degradation at a rate similar to wild-type p35 in cultured cell lines and primary neurons.
      Polyubiquitin works as a degradation signal for proteins targeted by proteasomes (
      • Kravtsova-Ivantsiv Y.
      • Ciechanover A.
      Non-canonical ubiquitin-based signals for proteasomal degradation.
      ,
      • Erales J.
      • Coffino P.
      Ubiquitin-independent proteasomal degradation.
      ). Thus, a question arises as to how proteasomes recognize and degrade p35 without the polyubiquitin tag. Some misfolded or impaired proteins are degraded without ubiquitination by default. There are several examples of proteins that display default degradation, although they are degraded physiologically in a ubiquitin-dependent manner. p53 is a tumor suppressor protein that is degraded by proteasomes via polyubiquitination by E3 ubiquitin ligases, such as Mdm2 (
      • Tsvetkov P.
      • Reuven N.
      • Shaul Y.
      Ubiquitin-independent p53 proteasomal degradation.
      ), but it is also degraded by the 20S proteasome by default if its N-terminal unstructured region is not protected by other proteins or modification. c-Fos proto-oncoprotein is an unstructured protein and degraded independently of ubiquitin by proteasomes when it does not form a transcriptional heterodimer with a partner protein (
      • Basbous J.
      • Jariel-Encontre I.
      • Gomard T.
      • Bossis G.
      • Piechaczyk M.
      Ubiquitin-independent versus ubiquitin-dependent proteasomal degradation of the c-Fos and Fra-1 trunscription factors: is there a unique answer?.
      ). p35 functions exclusively as the activator of Cdk5. Only a few p35 molecules exist as free p35 in vivo because Cdk5 is significantly more abundant than p35. If p35 fails to bind Cdk5, however, p35 would be recognized as a misfolded protein and degraded without ubiquitination by default. However, p35 23R appeared to maintain the proper conformation to fully bind and activate Cdk5, and it was degraded at a similar rate to that of wild-type p35. In this study we carried out most of the experiments under excess amounts of Cdk5 by co-expression. Thus, we think that it is unlikely that p35 23R is degraded through the default pathway of degradation.
      There are at least three types of substrate protein recognition by proteasomes in the ubiquitin-independent degradation systems, which are as follows: by the 19S regulatory particle of the 26S proteasome as an example of ornithine decarboxylase (
      • Coffino P.
      Antizyme, a mediator of ubiquitin-independent proteasomal degradation.
      ,
      • Takeuchi J.
      • Chen H.
      • Hoyt M.A.
      • Coffino P.
      Structural element of the ubiquitin-independent proteasomal degron of ornithine decarboxylase.
      ); by REGγ, also known as 11S or PA28, complexed with the 20S proteasome that is known for p21 Cdk inhibitor (
      • Chen X.
      • Barton L.F.
      • Chi Y.
      • Clurman B.E.
      • Roberts J.M.
      Ubiquitin-independent degradation of cell-cycle inhibitor by the REGgammma proteasome.
      ); by a core subunit of the 20S proteasome as exemplified by the F protein of hepatitis C virus (
      • Yuksek K.
      • Chen W.L.
      • Chien D.
      • Ou J.H.
      Ubiquitin-independent degradation of hepatitis C virus F protein.
      ). On the other hand, the amino acid sequence(s) required for degradation has also been investigated with several substrate proteins. In the case of the Rpn4 transcription factor that activates the expression of proteasome genes in yeast, the N-terminal unstructured segment and the following folded domain are essential for ubiquitin-independent degradation (
      • Ha S.W.
      • Ju D.
      • Xie Y.
      The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits.
      ). A similar requirement of two elements, including an unstructured region and a following α-helical sequence, is shown for thymidylate synthase (
      • Peña M.M.
      • Melo S.P.
      • Xing Y.Y.
      • White K.
      • Barbour K.W.
      • Berger F.G.
      The intrinsically disordered N-terminal domain of thymidylate synthase targets the enzyme to the ubiquitin-independent proteasomal degradation pathway.
      ). The two elements of thymidylate synthase function as a degradation signal if they are tagged at the C terminus of a reporter protein and called a ubiquitin-independent degron (
      • Melo S.P.
      • Barbour K.W.
      • Berger F.G.
      Cooperation between an intrinsically disordered region and a helical segment is required for ubiuquitin-independent degradation by the proteasome.
      ). According to the two-step model of degradation, the α-helical degron region is recognized by the proteasome, and then the disordered region enters into the proteasomal cavity. p35 may be degraded similarly because p35 has an unstructured ∼13-amino acids extension at the C terminus downstream of an α-helix-rich domain called the cyclin fold (Fig. 6D).
      NKX3.1 is a homeodomain transcription factor that regulates prostate cancer initiation and progression (
      • Guan B.
      • Pungaliya P.
      • Li X.
      • Uquillas C.
      • Mutton L.N.
      • Rubin E.H.
      • Bieberich C.J.
      Ubiquitination by TOPORS regulate the prostate tumor suppressor NKX3.1.
      ). NKX3.1 turnover is regulated by ubiquitination, but it is also proteolyzed by proteasomes independent of ubiquitination. This ubiquitin-independent degradation is mediated by a 21-amino acid sequence in its C-terminal region (
      • Rao V.
      • Guan B.
      • Mutton L.N.
      • Bieberich C.J.
      Proline-mediate proteasomal degradation of the prostate-specific tumor suppressor NKX3.1.
      ). The proline residue in the sequence is essential for its ubiquitin-independent degron activity. p35 has a homologous (∼53% identity) sequence at amino acids 240–258 with Pro-247 in a similar position (Fig. 6A). The mutation of Pro-247, which is in the ordered structure of the cyclin fold (
      • Tarricone C.
      • Dhavan R.
      • Peng J.
      • Areces L.B.
      • Tsai L.H.
      • Musacchio A.
      Structure and regulation of the CDK5-p25(nck5a) complex.
      ), to Ala slowed the turnover rate of p35 down remarkably. Considering that Pro-247 is positioned in the shallow concave (Fig. 6D), the structure, but not the amino acid sequence, around Pro-247 may provide the proteasome recognition site. Thus, p35 has two elements of an unstructured and a structured region next to each other that conform to the two-step degradation as shown by other proteins displaying ubiquitin-independent degradation.
      The physiological role of ubiquitin-independent degradation and its regulation are largely unknown for most proteins (
      • Kravtsova-Ivantsiv Y.
      • Ciechanover A.
      Non-canonical ubiquitin-based signals for proteasomal degradation.
      ,
      • Erales J.
      • Coffino P.
      Ubiquitin-independent proteasomal degradation.
      ). In the case of p21, however, it is indicated that the cell cycle-regulated degradation is ubiquitin-dependent (
      • Abbas T.
      • Sivaprasad U.
      • Terai K.
      • Amador V.
      • Pagano M.
      • Dutta A.
      PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex.
      ,
      • Bornstein G.
      • Bloom J.
      • Sitry-Shevah D.
      • Nakayama K.
      • Pagano M.
      • Hershko A.
      Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase.
      ), and its degradation during resting conditions is ubiquitin-independent (
      • Chen X.
      • Barton L.F.
      • Chi Y.
      • Clurman B.E.
      • Roberts J.M.
      Ubiquitin-independent degradation of cell-cycle inhibitor by the REGgammma proteasome.
      ,
      • Zhang H.
      • Cohen S.N.
      Smurf 2 up-regulation activates telomere-dependent senescence.
      ). Similar differential usage may be in operation for p35. p35 is unstable endogenously in neurons or when expressed heterogeneously in cultured cell lines, and p35 P247A showed a similar degradation rate to wild-type p35, suggesting that the degradation of p35 in resting neurons could be ubiquitin-independent. p35 is acutely degraded in neurons when treated with glutamate (
      • Wei F.Y.
      • Tomizawa K.
      • Ohshima T.
      • Asada A.
      • Saito T.
      • Nguyen C.
      • Bibb J.A.
      • Ishiguro K.
      • Kulkarni A.B.
      • Pant H.C.
      • Mikoshiba K.
      • Matsui H.
      • Hisanaga S.
      Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability.
      ,
      • Hosokawa T.
      • Saito T.
      • Asada A.
      • Ohshima T.
      • Itakura M.
      • Takahashi M.
      • Fukunaga K.
      • Hisanaga S.
      Enhanced activation of Ca2+/calmodulin-dependent protein kinase II upon down-regulation of cyclin-dependent kinase 5-p35.
      ). This stimulated degradation of p35 may be dependent on ubiquitination. In any case, rapid p35 turnover would be crucial for neurons to serve for their long life. Overactivation of Cdk5, which is induced by the p25 C-terminal stable fragment, is toxic for neurons. p25 is produced by the cleavage of p35 with calpain, and Cdk5 activated by p25 acquires a long lasting activity with free accessibility to proteins (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ), which Cdk5-p35 cannot access. The longer half-life of p35 may enhance the probability of the overproduction of p25. Two degradation pathways for p35 would be a mechanism to secure the long life of neurons.

      Author Contributions

      T. T. conceived the study and wrote the paper. S. M. designed, performed, and analyzed the experiments shown in FIGURE 1., FIGURE 2.. A. A. and T. S. provided technical assistance and performed a portion of the experiments shown in FIGURE 3., FIGURE 4., respectively. T. S. and H. K. provided technical assistance and contributed to the preparation of the figures. S. H. coordinated the study and wrote the paper. All of the authors reviewed the results and approved the final version of the manuscript.

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