Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain

Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/dephosphorylation to achieve experience-dependent alterations in brain circuitry. ERK is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro in intact mouse brain tissue and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr-292, whereas ERK and Cdk1 phosphorylate both Thr-292 and Thr-286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr-292 and Thr-286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of ∼5% or less. Phosphorylation of Thr-292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr-292 and Thr-286 MEK1 and that of ERK. Activation of the NMDA class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, whereas forced swim, a paradigm of acute stress, attenuates Thr-292 MEK1 phosphorylation. Together, the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission.     

A comparison of position-specific score matrices based on sequence and structure alignments

Sequence comparison methods based on position-specific score matrices (PSSMs) have proven a useful tool for recognition of the divergent members of a protein family and for annotation of functional sites. Here we investigate one of the factors that affects overall performance of PSSMs in a PSI-BLAST search, the algorithm used to construct the seed alignment upon which the PSSM is based. We compare PSSMs based on alignments constructed by global sequence similarity (ClustalW and ClustalW-pairwise), local sequence similarity (BLAST), and local structure similarity (VAST). To assess performance with respect to identification of conserved functional or structural sites, we examine the accuracy of the three-dimensional molecular models predicted by PSSM-sequence alignments. Using the known structures of those sequences as the standard of truth, we find that model accuracy varies with the algorithm used for seed alignment construction in the pattern local-structure (VAST) > local-sequence (BLAST) > global-sequence (ClustalW). Using structural similarity of query and database proteins as the standard of truth, we find that PSSM recognition sensitivity depends primarily on the diversity of the sequences included in the alignment, with an optimum around 30-50% average pairwise identity. We discuss these observations, and suggest a strategy for constructing seed alignments that optimize PSSM-sequence alignment accuracy and recognition sensitivity.     

Strategies for the effective identification of remotely related sequences in multiple PSSM search approach

Searches using position specific scoring matrices (PSSMs) have been commonly used in remote homology detection procedures such as PSI-BLAST and RPS-BLAST. A PSSM is generated typically using one of the sequences of a family as the reference sequence. In the case of PSI-BLAST searches the reference sequence is same as the query. Recently we have shown that searches against the database of multiple family-profiles, with each one of the members of the family used as a reference sequence, are more effective than searches against the classical database of single family-profiles. Despite relatively a better overall performance when compared with common sequence-profile matching procedures, searches against the multiple family-profiles database result in a few false positives and false negatives. Here we show that profile length and divergence of sequences used in the construction of a PSSM have major influence on the performance of multiple profile based search approach. We also identify that a simple parameter defined by the number of PSSMs corresponding to a family that is hit, for a query, divided by the total number of PSSMs in the family can distinguish effectively the true positives from the false positives in the multiple profiles search approach.     

Class 3 Semaphorin Mediates Dendrite Growth in Adult Newborn Neurons through Cdk5/FAK Pathway

Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP) 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397) and serine phosphorylation (Ser 732) of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.     

Use of multiple profiles corresponding to a sequence alignment enables effective detection of remote homologues

MOTIVATION: Position specific scoring matrices (PSSMs) corresponding to aligned sequences of homologous proteins are commonly used in homology detection. A PSSM is generated on the basis of one of the homologues as a reference sequence, which is the query in the case of PSI-BLAST searches. The reference sequence is chosen arbitrarily while generating PSSMs for reverse BLAST searches. In this work we demonstrate that the use of multiple PSSMs corresponding to a given alignment and variable reference sequences is more effective than using traditional single PSSMs and hidden Markov models. RESULTS: Searches for proteins with known 3-D structures have been made against three databases of protein family profiles corresponding to known structures: (1) One PSSM per family; (2) multiple PSSMs corresponding to an alignment and variable reference sequences for every family; and (3) hidden Markov models. A comparison of the performances of these three approaches suggests that the use of multiple PSSMs is most effective. CONTACT: ns@mbu.iisc.ernet.in.     

Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs

Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands. Each sequence motif is represented as a position-specific scoring matrix (PSSM) based on results from oriented peptide library and phage display experiments. Predicted domain-motif interactions from Scansite can be sequentially combined, allowing segments of biological pathways to be constructed in silico. The current release of Scansite, version 2.0, includes 62 motifs characterizing the binding and/or substrate specificities of many families of Ser/Thr- or Tyr-kinases, SH2, SH3, PDZ, 14-3-3 and PTB domains, together with signature motifs for PtdIns(3,4,5)P(3)-specific PH domains. Scansite 2.0 contains significant improvements to its original interface, including a number of new generalized user features and significantly enhanced performance. Searches of all SWISS-PROT, TrEMBL, Genpept and Ensembl protein database entries are now possible with run times reduced by approximately 60% when compared with Scansite version 1.0. Scansite 2.0 allows restricted searching of species-specific proteins, as well as isoelectric point and molecular weight sorting to facilitate comparison of predictions with results from two-dimensional gel electrophoresis experiments. Support for user-defined motifs has been increased, allowing easier input of user-defined matrices and permitting user-defined motifs to be combined with pre-compiled Scansite motifs for dual motif searching. In addition, a new series of Sequence Match programs for non-quantitative user-defined motifs has been implemented. Scansite is available via the World Wide Web at http://scansite.mit.edu.     

Cdk-mediated phosphorylation of the Kvβ2 auxiliary subunit regulates Kv1 channel axonal targeting

Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvβ2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)-mediated phosphorylation at these sites negatively regulates the interaction of Kvβ2 with the microtubule plus end-tracking protein EB1. Endogenous Cdks, EB1, and Kvβ2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvβ2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvβ2 to EB1.     

Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules

The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mouse brain but not in p35-/- mouse brain, suggesting that Cdk5/p39 is responsible for the in vivo phosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner.     

Identification of Cep169-interacting proteins and the in vivo modification sites of Cep169 via proteomic analysis

Cep169 is a microtubule plus-end tracking and centrosomal protein that interacts with CDK5RAP2. Cep169 is known to regulate microtubule dynamics and stability; however, its other cellular functions remain largely elusive. In this study, we identified novel Cep169-interacting proteins from HeLa cell extracts. Proteomic analysis via LC-MS/MS helped to identify approximately 400 novel Cep169-interacting proteins, including centrosomal proteins, cilium proteins, microtubule-associating proteins, and several E3 ubiquitin ligases. In addition, we identified in vivo posttranslational modification sites of Cep169, namely, 27 phosphorylation sites, five methylation sites, and four ubiquitination sites. Of these, 14 phosphorylated residues corresponding to the consensus Cdk phosphorylation sites may be required for Cdk1-mediated dissociation of Cep169 from the centrosome during mitosis and Cdk regulation during the G1/S phase. Furthermore, siRNA-induced Cep169 depletion was found to inhibit the growth of RPE1 cells. Our findings suggest that Cep169 regulates cell growth by interacting with multiple proteins.     

Cdk5 phosphorylation of EFhd2 at S74 affects its calcium binding activity

EFhd2 is a calcium binding protein, which is highly expressed in the central nervous system and associated with pathological forms of tau proteins in tauopathies. Previous phosphoproteomics studies and bioinformatics analysis suggest that EFhd2 may be phosphorylated. Here, we determine whether Cdk5, a hyperactivated kinase in tauopathies, phosphorylates EFhd2 and influence its known molecular activities. The results indicated that EFhd2 is phosphorylated by brain extract of the transgenic mouse CK-p25, which overexpresses the Cdk5 constitutive activator p25. Consistently, in vitro kinase assays demonstrated that Cdk5, but not GSK3β, directly phosphorylates EFhd2. Biomass, tandem mass spectrometry, and mutagenesis analyses indicated that Cdk5 monophosphorylates EFhd2 at S74, but not the adjacent S76. Furthermore, Cdk5-mediated phosphorylation of EFhd2 affected its calcium binding activity. Finally, a phospho-specific antibody was generated against EFhd2 phosphorylated at S74 and was used to detect this phosphorylation event in postmortem brain tissue from Alzheimer''s disease and normal-aging control cases. Results demonstrated that EFhd2 is phosphorylated in vivo at S74. These results imply that EFhd2''s physiological and/or pathological function could be regulated by its phosphorylation state.     

IMPALA: matching a protein sequence against a collection of PSI-BLAST-constructed position-specific score matrices

MOTIVATION: Many studies have shown that database searches using position-specific score matrices (PSSMs) or profiles as queries are more effective at identifying distant protein relationships than are searches that use simple sequences as queries. One popular program for constructing a PSSM and comparing it with a database of sequences is Position-Specific Iterated BLAST (PSI-BLAST). RESULTS: This paper describes a new software package, IMPALA, designed for the complementary procedure of comparing a single query sequence with a database of PSI-BLAST-generated PSSMs. We illustrate the use of IMPALA to search a database of PSSMs for protein folds, and one for protein domains involved in signal transduction. IMPALA's sensitivity to distant biological relationships is very similar to that of PSI-BLAST. However, IMPALA employs a more refined analysis of statistical significance and, unlike PSI-BLAST, guarantees the output of the optimal local alignment by using the rigorous Smith-Waterman algorithm. Also, it is considerably faster when run with a large database of PSSMs than is BLAST or PSI-BLAST when run against the complete non-redundant protein database.     

Design of accurate predictors for DNA-binding sites in proteins using hybrid SVM-PSSM method

In this paper, we investigate the design of accurate predictors for DNA-binding sites in proteins from amino acid sequences. As a result, we propose a hybrid method using support vector machine (SVM) in conjunction with evolutionary information of amino acid sequences in terms of their position-specific scoring matrices (PSSMs) for prediction of DNA-binding sites. Considering the numbers of binding and non-binding residues in proteins are significantly unequal, two additional weights as well as SVM parameters are analyzed and adopted to maximize net prediction (NP, an average of sensitivity and specificity) accuracy. To evaluate the generalization ability of the proposed method SVM-PSSM, a DNA-binding dataset PDC-59 consisting of 59 protein chains with low sequence identity on each other is additionally established. The SVM-based method using the same six-fold cross-validation procedure and PSSM features has NP=80.15% for the training dataset PDNA-62 and NP=69.54% for the test dataset PDC-59, which are much better than the existing neural network-based method by increasing the NP values for training and test accuracies up to 13.45% and 16.53%, respectively. Simulation results reveal that SVM-PSSM performs well in predicting DNA-binding sites of novel proteins from amino acid sequences.     

利用序列保守模体和局部构象信息预测转录因子结合位点

转录因子结合位点的计算预测是研究基因转录调控的重要环节,但常用的位置特异得分矩阵方法预测特异性偏低.通过深入分析结合位点的生物特征,提出了一种综合利用序列保守模体和局部构象信息的结合位点预测方法,以极大相关得分矩阵作为保守模体的描述模型,并根据二苷参数模型计算位点序列的局部构象,将两类信息得分组合为多维特征向量,在二次判别分析的框架下进行训练和滑动预测.预测过程中还引入了位置信息量以优化似然得分和过滤备选结果.针对大肠杆菌CRP和Fis结合位点数据的留一法测试结果表明,描述模型的改进和多种信息的融合能有效地改善预测方法的性能,大幅度提高特异性.     

Cyclin-dependent kinase 5 in neurofilament function and regulation

Neurofilaments are neuron-specific intermediate filaments. They are classed into three groups according to their molecular masses: neurofilament heavy, middle and light chains (NF-H, NF-M and NF-L). Neurofilaments assemble and form through the association of their central alpha-helical coiled-coil rod domains. NF-H and NF-M are distinct from NF-L as they contain a carboxyl-terminal tail domain, which appears to form connections with adjacent structures and other neurofilaments. Together with other axonal components such as microtubules, they form the dynamic axonal cytoskeleton. They maintain and regulate neuronal cytoskeletal plasticity through the regulation of neurite outgrowth, axonal caliber and axonal transport. Neurofilaments contain KSP repeats that are consensus motifs for the proline-directed kinases and are extensively phosphorylated in vivo, and their functions are thought to be regulated through their phosphorylation. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase, whose activity is restricted to the neuron through the neuronal-specific distribution of its activators p35 and p39. Cdk5 is the only kinase that affects the electrophoretic mobility of human NF-H and is thought to be the major neurofilament kinase. Cdk5 is involved in crosstalk with other signal transduction pathways such as the mitogen-activated protein kinase and myelin-associated glycoprotein pathways to influence the phosphorylation of neurofilaments and other cytoskeletal proteins. Both the hyperactivation of Cdk5 activity and subsequent hyperphosphorylation of neurofilaments and the microtubule-associated protein tau have been implicated in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. Here we review the functions of neurofilaments and the significance of Cdk5 phosphorylation of neurofilaments.     

Phosphorylation of the tubulin-binding protein, stathmin, by Cdk5 and MAP kinases in the brain

Regulation of cytoskeletal dynamics is essential to neuronal plasticity during development and adulthood. Dysregulation of these mechanisms may contribute to neuropsychiatric and neurodegenerative diseases. The neuronal protein kinase, cyclin-dependent kinase 5 (Cdk5), is involved in multiple aspects of neuronal function, including regulation of cytoskeleton. A neuroproteomic search identified the tubulin-binding protein, stathmin, as a novel Cdk5 substrate. Stathmin was phosphorylated by Cdk5 in vitro at Ser25 and Ser38, previously identified as mitogen-activated protein kinase (MAPK) and p38 MAPKdelta sites. Cdk5 predominantly phosphorylated Ser38, while MAPK and p38 MAPKdelta predominantly phosphorylated Ser25. Stathmin was phosphorylated at both sites in mouse brain, with higher levels in cortex and striatum. Cdk5 knockout mice exhibited decreased phospho-Ser38 levels. During development, phospho-Ser25 and -Ser38 levels peaked at post-natal day 7, followed by reduction in total stathmin. Inhibition of protein phosphatases in striatal slices caused an increase in phospho-Ser25 and a decrease in total stathmin. Interestingly, the prefrontal cortex of schizophrenic patients had increased phospho-Ser25 levels. In contrast, total and phospho-Ser25 stoichiometries were decreased in the hippocampus of Alzheimer's patients. Thus, microtubule regulatory mechanisms involving the phosphorylation of stathmin may contribute to developmental synaptic pruning and structural plasticity, and may be involved in neuropsychiatric and neurodegenerative disorders.     

Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr⁷²) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr⁷², we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr⁷² does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr⁷² regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.     

Phosphorylation of human DNA polymerase lambda by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen

DNA polymerase (Pol) λ is a member of the Pol X family and possesses four different enzymatic activities, being DNA polymerase, terminal transferase, deoxyribose phosphate lyase and polynucleotide synthetase, all localized in its C-terminal region. On the basis of its biochemical properties, Pol λ has been implicated in various DNA repair pathways, such as abasic site translesion DNA synthesis, base excision repair and non-homologous end joining of double strand breaks. However, its role in vivo has not yet been elucidated. In addition, Pol λ has been shown to interact with the replication clamp proliferating cell nuclear antigen (PCNA) in vitro and in vivo. In this work, we searched by affinity chromatography for novel partners and we identified the cyclin-dependent kinase Cdk2 as novel partner of Pol λ. Pol λ is phosphorylated in vitro by several Cdk/cyclin complexes, including Cdk2/cyclin A, in its proline-serine-rich domain. While the polymerase activity of Pol λ was not affected by Cdk2/cyclin A phosphorylation, phosphorylation of Pol λ was decreased by its interaction with PCNA. Finally, Pol λ is also phosphorylated in vivo in human cells and this phosphorylation is modulated during the cell cycle.     

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

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 (1, 2). 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) (3, 4). Phosphorylation site-specific antibodies are frequently used to detect phosphorylation at target sites (5, 6). 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 (7–10), 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 (11). 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 (12–21).Cyclin-dependent kinase 5 (Cdk5)¹ 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 (22–24). Cdk5 is activated by binding to activation subunit p35 and inactivated by proteasomal degradation of p35 (25). In addition, Cdk5 activity is deregulated by cleavage of p35 to p25 with calpain, resulting in abnormal activation and ultimately causing neuronal cell death (26–29). Proteolysis of p35, either by proteasomal degradation or cleavage by calpain, is regulated by phosphorylation of p35 by Cdk5 (30–33). 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 (33). We also showed that phosphorylation of p35 decreased during brain development and proposed its relationship to age-dependent vulnerability of neurons to stress stimuli (32). 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.