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Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE*

  • Tomohisa Hosokawa
    Correspondence
    To whom correspondence may be addressed. Tel.: 81-42-677-2754; Fax: 81-42-677-2559;
    Affiliations
    Department of Biological Sciences, Graduate School of Science and Technology, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan and
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  • Taro Saito
    Affiliations
    Department of Biological Sciences, Graduate School of Science and Technology, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan and
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  • Akiko Asada
    Affiliations
    Department of Biological Sciences, Graduate School of Science and Technology, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan and
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  • Kohji Fukunaga
    Affiliations
    Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan
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  • Shin-ichi Hisanaga
    Correspondence
    To whom correspondence may be addressed. Tel.: 81-42-677-2754; Fax: 81-42-677-2559;
    Affiliations
    Department of Biological Sciences, Graduate School of Science and Technology, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan and
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  • Author Footnotes
    * This work was supported by a grant-in-aid from the Japan Society for the Promotion of Science (to T. H.) and a grant-in-aid from the Ministry of Education, Culture, Sports, and Science and Technology of Japan (to S. H.).
    This article contains supplemental Figs. S1–S3.
Open AccessPublished:January 23, 2010DOI:https://doi.org/10.1074/mcp.M900578-MCP200
      Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser8 and Thr138 as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser91, which was phosphorylated by Ca2+-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser8, Ser91, and Thr138 phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser8 was phosphorylated in all p35 molecules, whereas Ser91 was phosphorylated in 60% and Thr138 was phosphorylated in ∼12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.
      Phosphorylation is a major post-translational modification of proteins, modulating a variety of cellular functions (
      • Hunter T.
      Signaling—2000 and beyond.
      ,
      • Cohen P.
      Protein kinases—the major drug targets of the twenty-first century?.
      ). Because most phosphorylation occurs in a highly site-specific manner, identification of phosphorylation sites has been a subject of intense investigation. Several analytical methods have been utilized to identify phosphorylation sites, including mass spectrometry, amino acid sequencing, and radioisotope phosphate labeling of proteins with mutation(s) at putative phosphorylation site(s) (
      • Thingholm T.E.
      • Jensen O.N.
      • Larsen M.R.
      Analytical strategies for phosphoproteomics.
      ,
      • Kalume D.E.
      • Molina H.
      • Pandey A.
      Tackling the phosphoproteome: tools and strategies.
      ). Phosphorylation site-specific antibodies are frequently used to detect phosphorylation at target sites (
      • Inagaki M.
      • Inagaki N.
      • Takahashi T.
      • Takai Y.
      Phosphorylation-dependent control of structures of intermediate filaments: a novel approach using site- and phosphorylation state-specific antibodies.
      ,
      • Kaufmann H.
      • Bailey J.E.
      • Fussenegger M.
      Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis.
      ). Many phosphospecific antibodies are now commercially available. These phosphospecific antibodies are convenient and useful tools for examining site-specific phosphorylation both in vivo and in vitro. However, they are not appropriate for estimating quantitative ratios of phosphorylation states. Electrophoretic mobility shift on SDS-PAGE is also often used to observe phosphorylation (
      • Wegener A.D.
      • Jones L.R.
      Phosphorylation-induced mobility shift in phospholamban in sodium dodecyl sulfate-polyacrylamide gels. Evidence for a protein structure consisting of multiple identical phosphorylatable subunits.
      ,
      • Baudier J.
      • Cole R.D.
      Phosphorylation of tau proteins to a state like that in Alzheimer's brain is catalyzed by a calcium/calmodulin-dependent kinase and modulated by phospholipids.
      ,
      • Morishima M.
      • Ihara Y.
      Posttranslational modifications of tau in paired helical filaments.
      ,
      • Hisanaga S.
      • Kusubata M.
      • Okumura E.
      • Kishimoto T.
      Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules.
      ), but this method is not always applied to site-specific phosphorylation.
      Phos-tag is a newly developed dinuclear metal complex that can be used to provide phosphate-binding sites when conjugated to analytical materials such as acrylamide and biotin (
      • Kinoshita E.
      • Kinoshita-Kikuta E.
      • Takiyama K.
      • Koike T.
      Phosphate-binding tag, a new tool to visualize phosphorylated proteins.
      ). In SDS-PAGE using Phos-tag acrylamide, phosphorylated proteins are trapped by the Phos-tag sites, delaying their migration and thus separating them from unphosphorylated proteins. Subsequent immunoblot analysis with phosphorylation-independent antibodies reveals both the phosphorylated and unphosphorylated bands. Because the migration of the phosphorylated proteins is greatly delayed compared with migration in Laemmli SDS-PAGE, it is easy to identify the phosphorylated proteins from observed positions on blots. In the past 3 years, this method has been used to detect phosphorylation states for many proteins such as ERK1/2, cdc37, myosin light chain, eIF2α, protein kinase D, β-casein, SIRT7, and dysbindin-1 (
      • Kinoshita-Kikuta E.
      • Aoki Y.
      • Kinoshita E.
      • Koike T.
      Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis.
      ,
      • Miyata Y.
      • Nishida E.
      Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis.
      ,
      • Oh H.
      • Irvine K.D.
      In vivo regulation of Yorkie phosphorylation and localization.
      ,
      • Takeya K.
      • Loutzenhiser K.
      • Shiraishi M.
      • Loutzenhiser R.
      • Walsh M.P.
      A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles.
      ,
      • Tatematsu K.
      • Yoshimoto N.
      • Okajima T.
      • Tanizawa K.
      • Kuroda S.
      Identification of ubiquitin ligase activity of RBCK1 and its inhibition by splice variant RBCK2 and protein kinase Cbeta.
      ,
      • Igarashi J.
      • Murase M.
      • Iizuka A.
      • Pichierri F.
      • Martinkova M.
      • Shimizu T.
      Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins.
      ,
      • Sumara G.
      • Formentini I.
      • Collins S.
      • Sumara I.
      • Windak R.
      • Bodenmiller B.
      • Ramracheya R.
      • Caille D.
      • Jiang H.
      • Platt K.A.
      • Meda P.
      • Aebersold R.
      • Rorsman P.
      • Ricci R.
      Regulation of PKD by the MAPK p38delta in insulin secretion and glucose homeostasis.
      ,
      • Kinoshita E.
      • Kinoshita-Kikuta E.
      • Matsubara M.
      • Aoki Y.
      • Ohie S.
      • Mouri Y.
      • Koike T.
      Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.
      ,
      • Grob A.
      • Roussel P.
      • Wright J.E.
      • McStay B.
      • Hernandez-Verdun D.
      • Sirri V.
      Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.
      ,
      • Oyama S.
      • Yamakawa H.
      • Sasagawa N.
      • Hosoi Y.
      • Futai E.
      • Ishiura S.
      Dysbindin-1, a schizophrenia-related protein, functionally interacts with the DNA-dependent protein kinase complex in an isoform-dependent manner.
      ).
      Cyclin-dependent kinase 5 (Cdk5)
      The abbreviations used are:
      Cdk
      cyclin-dependent kinase
      Phos-tag
      phosphate-binding tag
      NMDA
      N-methyl-d-aspartate
      BAP
      bacterial alkaline phosphatase
      CaMKII
      Ca2+-calmodulin-dependent protein kinase II
      ERK
      extracellular signal-regulated kinase
      P
      postnatal day
      E
      embryonic day
      DIV
      days in vitro
      kn
      kinase-negative
      WT
      wild-type
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      Ca2+-CaM
      calcium and calmodulin.
      1The abbreviations used are:Cdk
      cyclin-dependent kinase
      Phos-tag
      phosphate-binding tag
      NMDA
      N-methyl-d-aspartate
      BAP
      bacterial alkaline phosphatase
      CaMKII
      Ca2+-calmodulin-dependent protein kinase II
      ERK
      extracellular signal-regulated kinase
      P
      postnatal day
      E
      embryonic day
      DIV
      days in vitro
      kn
      kinase-negative
      WT
      wild-type
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      Ca2+-CaM
      calcium and calmodulin.
      is a proline-directed serine/threonine kinase that is expressed predominantly in postmitotic neurons and regulates various neuronal events such as neuronal migration, spine formation, synaptic activity, and cell death (
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ,
      • Cheung Z.H.
      • Ip N.Y.
      The roles of cyclin-dependent kinase 5 in dendrite and synapse development.
      ,
      • Lim A.C.
      • Qi R.Z.
      Cyclin-dependent kinases in neural development and degeneration.
      ). Cdk5 is activated by binding to activation subunit p35 and inactivated by proteasomal degradation of p35 (
      • Hisanaga S.
      • Saito T.
      The regulation of cyclin-dependent kinase 5 activity through the metabolism of p35 or p39 Cdk5 activator.
      ). In addition, Cdk5 activity is deregulated by cleavage of p35 to p25 with calpain, resulting in abnormal activation and ultimately causing neuronal cell death (
      • 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.
      ,
      • Saito T.
      • Konno T.
      • Hosokawa T.
      • Asada A.
      • Ishiguro K.
      • Hisanaga S.
      p25/cyclin-dependent kinase 5 promotes the progression of cell death in nucleus of endoplasmic reticulum-stressed neurons.
      ). Proteolysis of p35, either by proteasomal degradation or cleavage by calpain, is regulated by phosphorylation of p35 by Cdk5 (
      • 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.
      ,
      • Saito T.
      • Onuki R.
      • Fujita Y.
      • Kusakawa G.
      • Ishiguro K.
      • Bibb J.A.
      • Kishimoto T.
      • Hisanaga S.
      Developmental regulation of the proteolysis of the p35 cyclin-dependent kinase 5 activator by phosphorylation.
      ,
      • Kamei H.
      • Saito T.
      • Ozawa M.
      • Fujita Y.
      • Asada A.
      • Bibb J.A.
      • Saido T.C.
      • Sorimachi H.
      • Hisanaga S.
      Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.
      ). Therefore, phosphorylation of p35 is essential for proper regulation of Cdk5 activity and function. We previously identified Ser8 and Thr138 as major p35 phosphorylation sites (
      • Kamei H.
      • Saito T.
      • Ozawa M.
      • Fujita Y.
      • Asada A.
      • Bibb J.A.
      • Saido T.C.
      • Sorimachi H.
      • Hisanaga S.
      Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.
      ). We also showed that phosphorylation of p35 decreased during brain development and proposed its relationship to age-dependent vulnerability of neurons to stress stimuli (
      • Saito T.
      • Onuki R.
      • Fujita Y.
      • Kusakawa G.
      • Ishiguro K.
      • Bibb J.A.
      • Kishimoto T.
      • Hisanaga S.
      Developmental regulation of the proteolysis of the p35 cyclin-dependent kinase 5 activator by phosphorylation.
      ). Thus, to understand the in vivo regulation of Cdk5 activity, it is critical to analyze the phosphorylation states of p35 in brain. However, there is no convenient method to analyze the precise in vivo phosphorylation status of the endogenous proteins.
      In this study, we applied the Phos-tag SDS-PAGE method to analyze the phosphorylation states of p35 in vivo and in cultured neurons. We constructed standard band profiles of phosphorylated p35 by Phos-tag SDS-PAGE using Ala mutants at Ser8 and/or Thr138. From these experiments, we observed an unidentified in vivo phosphorylation site at Ser91. We quantified the phosphorylation at each site in cultured neurons and brain, providing the first quantitative estimate of the in vivo phosphorylation states of p35. We discuss the usefulness of Phos-tag SDS-PAGE to analyze the in vivo phosphorylation states of proteins.

      DISCUSSION

      In this study, we analyzed the in vivo phosphorylation states of a p35 Cdk5 activator quantitatively using Phos-tag SDS-PAGE followed by immunoblotting with a phosphorylation-independent antibody to p35. p35 displayed phosphorylation site-dependent upward shifts in electrophoretic mobility on Phos-tag SDS-PAGE that enabled us to identify the phosphorylation states of p35 without using radioisotope or phosphospecific antibodies. After establishing the standard phosphorylation profile of p35, we evaluated the in vivo phosphorylation states of p35 in cultured neurons and mouse brain. In fetal mouse brain, for example, Ser8 was phosphorylated in all p35 molecules, and Thr138 was phosphorylated in 20% of p35 molecules. In addition, we identified Ser91 as a novel in vivo phosphorylation site that was phosphorylated by CaMKII in vitro and was phosphorylated in about 60% of p35 molecules in fetal mouse brain. The overall phosphorylation at each of these residues was reduced in adult mouse brain. Because Cdk5 activity is regulated by phosphorylation-dependent proteolysis of p35, the quantitative analysis of in vivo phosphorylation states of p35 will be useful for exploring in vivo regulation of Cdk5 activity. Our analysis also demonstrates the general usefulness of Phos-tag SDS-PAGE as a method for in vivo phosphorylation analysis of proteins.
      Phos-tag SDS-PAGE is a recently developed method that is capable of separating phosphorylated proteins on SDS-PAGE (
      • Kinoshita-Kikuta E.
      • Aoki Y.
      • Kinoshita E.
      • Koike T.
      Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis.
      ) and has been used to show phosphorylation of proteins (
      • Kinoshita-Kikuta E.
      • Aoki Y.
      • Kinoshita E.
      • Koike T.
      Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis.
      ,
      • Miyata Y.
      • Nishida E.
      Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis.
      ,
      • Oh H.
      • Irvine K.D.
      In vivo regulation of Yorkie phosphorylation and localization.
      ,
      • Takeya K.
      • Loutzenhiser K.
      • Shiraishi M.
      • Loutzenhiser R.
      • Walsh M.P.
      A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles.
      ,
      • Tatematsu K.
      • Yoshimoto N.
      • Okajima T.
      • Tanizawa K.
      • Kuroda S.
      Identification of ubiquitin ligase activity of RBCK1 and its inhibition by splice variant RBCK2 and protein kinase Cbeta.
      ,
      • Igarashi J.
      • Murase M.
      • Iizuka A.
      • Pichierri F.
      • Martinkova M.
      • Shimizu T.
      Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins.
      ,
      • Sumara G.
      • Formentini I.
      • Collins S.
      • Sumara I.
      • Windak R.
      • Bodenmiller B.
      • Ramracheya R.
      • Caille D.
      • Jiang H.
      • Platt K.A.
      • Meda P.
      • Aebersold R.
      • Rorsman P.
      • Ricci R.
      Regulation of PKD by the MAPK p38delta in insulin secretion and glucose homeostasis.
      ,
      • Kinoshita E.
      • Kinoshita-Kikuta E.
      • Matsubara M.
      • Aoki Y.
      • Ohie S.
      • Mouri Y.
      • Koike T.
      Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes.
      ,
      • Grob A.
      • Roussel P.
      • Wright J.E.
      • McStay B.
      • Hernandez-Verdun D.
      • Sirri V.
      Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.
      ,
      • Oyama S.
      • Yamakawa H.
      • Sasagawa N.
      • Hosoi Y.
      • Futai E.
      • Ishiura S.
      Dysbindin-1, a schizophrenia-related protein, functionally interacts with the DNA-dependent protein kinase complex in an isoform-dependent manner.
      ). When this technique was initially reported, Kinoshita et al. (
      • Kinoshita-Kikuta E.
      • Aoki Y.
      • Kinoshita E.
      • Koike T.
      Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis.
      ) predicted its use in quantitative estimation of in vivo phosphorylation. However, most previous reports used this technique qualitatively as one of several methods indicating phosphorylation of particular proteins. A quantitative application of Phos-tag SDS-PAGE was reported with myosin light chain, but site-specific quantification was not addressed (
      • Takeya K.
      • Loutzenhiser K.
      • Shiraishi M.
      • Loutzenhiser R.
      • Walsh M.P.
      A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles.
      ). We report here that the upward mobility shifts differed among the phosphorylation sites in p35 where the largest upward shift of phosphorylation was at Ser91 followed by Ser8 and finally Thr138, which was a small but distinct shift. Although the chemical basis for phosphorylation site-specific mobility variation is not known, we were able to use this method successfully to identify site-specific phosphorylation-dependent shifts.
      Using two-dimensional phosphopeptide mapping and anti-phosphoantibody analysis, we previously reported that p35 is phosphorylated at Ser8 and Thr138 in COS-7 cells and at Ser8 in cultured neurons (
      • Kamei H.
      • Saito T.
      • Ozawa M.
      • Fujita Y.
      • Asada A.
      • Bibb J.A.
      • Saido T.C.
      • Sorimachi H.
      • Hisanaga S.
      Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.
      ). Furthermore, we showed that phosphorylation decreases in developing rat brain (
      • Saito T.
      • Onuki R.
      • Fujita Y.
      • Kusakawa G.
      • Ishiguro K.
      • Bibb J.A.
      • Kishimoto T.
      • Hisanaga S.
      Developmental regulation of the proteolysis of the p35 cyclin-dependent kinase 5 activator by phosphorylation.
      ). However, we did not have a method to address quantitative phosphorylation of endogenous p35 in cultured neurons or brain at that time. The quantitative measurement obtained here supports the previous qualitative results and further demonstrates that there are differences in the extent of phosphorylation between transfected cells, primary cultured neurons, and brain cells in vivo. Ser8 was phosphorylated in most p35 molecules whether they were exogenously expressed in transfected COS-7 cells or endogenously expressed in primary cultured neurons and embryonic brain. The phosphorylation of Thr138 was detected most extensively in COS-7 cells (91% of p35 molecules) followed by primary neurons (40%) and embryonic brain (<12%). In contrast, the phosphorylation of Ser91 was least extensive in COS-7 cells (9%) followed by primary neurons (21%) and embryonic brain (59%). These results suggest that phosphorylation of ectopically expressed proteins is not always identical to that of proteins in endogenous tissues.
      The phosphorylation of Ser8 was found in p35 co-expressed with Cdk5 but not with knCdk5. Thus, Ser8 was phosphorylated by Cdk5. In fetal brain, Ser8 was phosphorylated in all p35 molecules. This result was somewhat surprising even though Ser8 is autophosphorylated. This high phosphorylation level is not due to its inability to dephosphorylate after it has been once phosphorylated. Ser8 was dephosphorylated by incubation of brain extract in the absence of phosphatase inhibitors. Ser8 was strongly phosphorylated over a short time during metabolic labeling experiments of cultured neurons (
      • Kamei H.
      • Saito T.
      • Ozawa M.
      • Fujita Y.
      • Asada A.
      • Bibb J.A.
      • Saido T.C.
      • Sorimachi H.
      • Hisanaga S.
      Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.
      ). These results suggest that phosphorylation of Ser8 is maintained by continuous phosphorylation even after it is dephosphorylated. Continuous phosphorylation may be accomplished by intramolecular phosphorylation in the active Cdk5-p35 complex. In contrast, phosphorylation of Thr138 was not the case. We hypothesized that Thr138 could be the phosphorylation site for Cdk5 because Thr138 is in the TPKR sequence, the most preferred consensus sequence for Cdk5 (
      • Kesavapany S.
      • Li B.S.
      • Amin N.
      • Zheng Y.L.
      • Grant P.
      • Pant H.C.
      Neuronal cyclin-dependent kinase 5: role in nervous system function and its specific inhibition by the Cdk5 inhibitory peptide.
      ). This possibility is supported by the observation that co-expression of p35 with Cdk5 enhanced phosphorylation of Thr138. However, this site was also phosphorylated when p35 was co-expressed with knCdk5 in COS-7 cells. Cycling Cdks such as Cdk1 and Cdk2, whose substrate specificity is similar to Cdk5 (
      • Hisanaga S.
      • Uchiyama M.
      • Hosoi T.
      • Yamada K.
      • Honma N.
      • Ishiguro K.
      • Uchida T.
      • Dahl D.
      • Ohsumi K.
      • Kishimoto T.
      Porcine brain neurofilament-H tail domain kinase: its identification as cdk5/p26 complex and comparison with cdc2/cyclin B kinase.
      ), are potential candidates for phosphorylating Thr138 in COS-7 cells. These results indicate that Thr138 can be phosphorylated by other proline-directed kinases and that, as opposed to Ser8, Thr138 phosphorylation would be intermolecular even when the site is phosphorylated by Cdk5.
      In addition to Ser8 and Thr138, several unidentified minor phosphorylation sites were detected in p35 expressed in COS-7 cells using Phos-tag SDS-PAGE analysis. For the site designated X1, phosphorylation shifted the 2A mutant to the L3 position by Phos-tag SDS-PAGE when co-expressed with Cdk5. Because this phosphorylation was observed in the 2A mutant by co-expression with Cdk5 but not with knCdk5, this site could be one of the other p35 (S/T)P sites, Ser170 and Thr197 (Fig. 2C). This is consistent with the recent report by He et al. (
      • He L.
      • Hou Z.
      • Qi R.Z.
      Calmodulin binding and Cdk5 phosphorylation of p35 regulate its effect on microtubules.
      ) that the 2A mutant co-expressed with Cdk5 in HEK293 cells is still phosphorylated to some extent. However, this phosphorylation appeared only to a small extent, if at all, in neurons of mouse brain because the bands that could possibly include phosphorylated X1 were not clearly detectable in p35 prepared from brain. Therefore, in this study, we did not attempt to determine whether Ser170 or Thr197 corresponds to the X1 site. Another novel phosphorylation site named X2 became obvious when the immunoblot using antibody C19 (anti-p35) was overexposed. This band was detected even in the 2A mutant co-expressed with knCdk5, indicating that the site was phosphorylated at an amino acid other than the (S/T)P sites by a different kinase. The finding that phosphorylation at this site was detected in endogenous p35 in neurons prompted us to identify the X2 site (see below). These two phosphorylation sites were not evident in our previous analysis by two-dimensional phosphopeptide mapping following metabolic labeling of neurons or COS-7 cells with [32P]phosphate (
      • Kamei H.
      • Saito T.
      • Ozawa M.
      • Fujita Y.
      • Asada A.
      • Bibb J.A.
      • Saido T.C.
      • Sorimachi H.
      • Hisanaga S.
      Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.
      ). Lower levels of phosphorylation and/or a slower turnover rate may explain why the X1 and X2 sites were not identified in earlier studies. In either case, Phos-tag SDS-PAGE can be applied to detect minor phosphorylation sites as well as those with a slow turnover rate.
      Because phosphorylation of X2 increased upon NMDA treatment, which activates CaMKII (
      • Bliss T.V.
      • Collingridge G.L.
      A synaptic model of memory: long-term potentiation in the hippocampus.
      ), we hypothesized that the X2 site could be a CaMKII phosphorylation site. Using Ala mutants at CaMKII consensus (R/K)XX(S/T) phosphorylation sites, the X2 site was identified as Ser91. We confirmed the phosphorylation of Ser91 in fetal and adult brains using phosphospecific antibody (Ser(P)91) generated in this study. Mass spectrometric analysis may be an alternative way to confirm this phosphorylation, although this might not be easy for p35 because only a small percentage of p35, that is a small amount of unstable protein, was phosphorylated at Ser91 in adult brains. In general, however, combinatory use of mass spectroscopy to map phosphorylation sites after identifying phosphorylation using Phos-tag SDS-PAGE will be a powerful biochemical analytic tool.
      Although we cannot conclude that Ser91 is phosphorylated exclusively by CaMKII in vivo, based on published results that a population of p35 localizes in the postsynaptic region (
      • 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 downregulation of cyclin-dependent kinase 5-p35.
      ,
      • Lai K.O.
      • Ip N.Y.
      Recent advances in understanding the roles of Cdk5 in synaptic plasticity.
      ) and associates with CaMKII (
      • Dhavan R.
      • Greer P.L.
      • Morabito M.A.
      • Orlando L.R.
      • Tsai L.H.
      The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner.
      ), it is likely that CaMKII phosphorylates p35 at Ser91 in neurons. Indeed, the phosphorylation of Ser91 was lower in COS-7 cells than in primary neurons and fetal brain, reflecting expression levels of CaMKII, which are abundant in neuronal cells compared with rat kidney cells (
      • Tobimatsu T.
      • Fujisawa H.
      Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs.
      ). Ser91 phosphorylation was reduced considerably with development, dropping from 59% in embryonic brain to 8% in adult brain. This result may simply reflect the decreased activation frequency of CaMKII in adult mouse brain. In fact, it is reported that active CaMKII decreases during brain development (
      • Molloy S.S.
      • Kennedy M.B.
      Autophosphorylation of type II Ca2+/calmodulin-dependent protein kinase in cultures of postnatal rat hippocampal slices.
      ,
      • Yamamoto H.
      • Hiragami Y.
      • Murayama M.
      • Ishizuka K.
      • Kawahara M.
      • Takashima A.
      Phosphorylation of tau at serine 416 by Ca2+/calmodulin-dependent protein kinase II in neuronal soma in brain.
      ). On the other hand, we reported previously that Cdk5 activity suppresses CaMKII activation (
      • 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 downregulation of cyclin-dependent kinase 5-p35.
      ). Thus, the interaction between Cdk5 and CaMKII is bidirectional. CaMKII is a well known mediator of long term potentiation (
      • Thiel G.
      • Czernik A.J.
      • Gorelick F.
      • Nairn A.C.
      • Greengard P.
      Ca2+/calmodulin-dependent protein kinase II: identification of threonine-286 as the autophosphorylation site in the alpha subunit associated with the generation of Ca2+-independent activity.
      ,
      • Fukunaga K.
      • Stoppini L.
      • Miyamoto E.
      • Muller D.
      Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase II.
      ), and Cdk5 has also been shown to be involved in memory formation (
      • Lai K.O.
      • Ip N.Y.
      Recent advances in understanding the roles of Cdk5 in synaptic plasticity.
      ,
      • 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.
      ,
      • Ohshima T.
      • Ogura H.
      • Tomizawa K.
      • Hayashi K.
      • Suzuki H.
      • Saito T.
      • Kamei H.
      • Nishi A.
      • Bibb J.A.
      • Hisanaga S.
      • Matsui H.
      • Mikoshiba K.
      Impairment of hippocampal long-term depression and defective spatial learning and memory in p35 mice.
      ,
      • Hawasli A.H.
      • Benavides D.R.
      • Nguyen C.
      • Kansy J.W.
      • Hayashi K.
      • Chambon P.
      • Greengard P.
      • Powell C.M.
      • Cooper D.C.
      • Bibb J.A.
      Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.
      ,
      • Sananbenesi F.
      • Fischer A.
      • Wang X.
      • Schrick C.
      • Neve R.
      • Radulovic J.
      • Tsai L.H.
      A hippocampal Cdk5 pathway regulates extinction of contextual fear.
      ). The cross-talk between these molecules would be an important regulatory mechanism in synaptic plasticity.
      In summary, we used Phos-tag SDS-PAGE for the first time to estimate site-specific in vivo phosphorylation of p35. Quantification of site-specific phosphorylation can be investigated by other means, for example by stable isotope mass spectrometry using multiple reaction monitoring (
      • Mayya V.
      • Rezual K.
      • Wu L.
      • Fong M.B.
      • Han D.K.
      Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry: determination of inhibitory phosphorylation status of cyclin-dependent kinases.
      ,
      • Wolf-Yadlin A.
      • Hautaniemi S.
      • Lauffenburger D.A.
      • White F.M.
      Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks.
      ,
      • Ciccimaro E.
      • Hanks S.K.
      • Yu K.H.
      • Blair I.A.
      Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry.
      ). However, the number of studies using this method has been limited. We successfully used the Phos-tag SDS-PAGE method to determine populations of p35 with different combinations of phosphorylation sites. The successful use of the method is due to our previous determination of two major phosphorylation sites in p35 and the limited number of phosphorylation sites. We note that this method has advantages over other methods that have been used to analyze phosphorylation of proteins. This method does not require special equipment other than that used for typical electrophoresis and blotting, nor does it require radioisotope labeling, purification of the proteins, or a catalogue of site-specific phosphorylation-dependent antibodies. Lysates of tissues or cells can be directly subjected to this method followed by immunoblotting with a phosphorylation-independent antibody, although prior identification of phosphorylation sites of target proteins would be preferable to maximally utilize this method. We also have to note the negative aspects of this method. This method takes more time than mass spectroscopy and requires reliable antibody for quantification. Nevertheless, we believe that this method is a simple and convenient way to quantify the in vivo phosphorylation states of proteins.

      Acknowledgments

      We thank Dr. Masaki Inagaki (Aichi Cancer Center Research Institute, Nagoya, Japan) for helpful advice about generating the phosphospecific antibody. We also thank Dr. Junjiro Horiuchi (Tokyo Metropolitan University, Tokyo, Japan) for reading the manuscript.

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