Identification and structure characterization of a Cdk inhibitory peptide derived from neuronal-specific Cdk5 activator

The activation of cyclin-dependent kinase 5 (Cdk5) depends on the binding of its neuronal specific activator Nck5a. The minimal activation domain of Nck5a is located in the region of amino acid residues 150 to 291 (Tang, D., Chun, A. C. S., Zhang, M., and Wang, J. H. (1997) J. Biol. Chem. 272, 12318-12327). In this work we show that a 29-residue peptide, denoted as the alphaN peptide, encompassing amino acid residues Gln145 to Asp173 of Nck5a is capable of binding Cdk5 to result in kinase inhibition. This peptide also inhibits an active phospho-Cdk2-cyclin A complex, with a similar potency. Direct competition experiments have shown that this inhibitory peptide does not compete with Nck5a or cyclin A for Cdk5 or Cdk2, respectively. Steady state kinetic analysis has indicated that the alphaN peptide acts as a non-competitive inhibitor of Cdk5. Nck5a complex with respect to the peptide substrate. To understand the molecular basis of kinase inhibition by the peptide, we determined the structure of the peptide in solution by circular dichroism and two-dimensional 1H NMR spectroscopy. The peptide adopts an amphipathic alpha-helical structure from residues Ser149 to Arg162 which can be further stabilized by the helix-stabilizing solvent trifluoroethanol. The hydrophobic face of the helix is likely to be the kinase binding surface.     

Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor

Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.     

Identification of a common protein association region in the neuronal Cdk5 activator

Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.     

Protein kinase CK2 is an inhibitor of the neuronal Cdk5 kinase

The complex of Cdk5 and its neuronal activator p35 is a proline-directed Ser/Thr kinase that plays an important role in various neuronal functions. Deregulation of the Cdk5 enzymatic activity was found to associate with a number of neurodegenerative diseases. To search for regulatory factors of Cdk5-p35 in the brain, we developed biochemical affinity isolation using a recombinant protein comprising the N-terminal 149 amino acids of p35. The catalytic alpha-subunit of protein kinase CK2 (formerly known as casein kinase 2) was identified by mass spectrometry from the isolation. The association of CK2 with p35 and Cdk5 was demonstrated, and the CK2-binding sites were delineated in p35. Furthermore, CK2 displayed strong inhibition toward the Cdk5 activation by p35. The Cdk5 inhibition is dissociated from the kinase function of CK2 because the kinase-dead mutant of CK2 displayed the similar Cdk5 inhibitory activity as the wild-type enzyme. Further characterization showed that CK2 blocks the complex formation of Cdk5 and p35. Together, these findings suggest that CK2 acts as an inhibitor of Cdk5 in the brain.     

The protein kinase Cdk5. Structural aspects, roles in neurogenesis and involvement in Alzheimer's pathology.

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3beta (GSK3 beta), MARK kinase, MAP kinase, the cyclin-dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron-specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule-asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin-folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline-directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C-terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the beta-amyloid peptide (A beta) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau-phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by A beta. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by A beta in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.     

DNA binding protein dbpA binds Cdk5 and inhibits its activity

Progress in the cell cycle is governed by the activity of cyclin dependent kinases (Cdks). Unlike other Cdks, the Cdk5 catalytic subunit is found mostly in differentiated neurons. Interestingly, the only known protein that activates Cdk5 (i.e. p35) is expressed solely in the brain. It has been suggested that, besides its requirement in neuronal differentiation, Cdk5 activity is induced during myogenesis. However, it is not clear how this activity is regulated in the pathway that leads proliferative cells to differentiation. In order to find if there exists any Cdk5-interacting protein, the yeast two-hybrid system was used to screen a HeLa cDNA library. We have determined that a C-terminal 172 amino acid domain of the DNA binding protein, dbpA, binds to Cdk5. Biochemical analyses reveal that this fragment (dbpA(Cdelta)) strongly inhibits p35-activated Cdk5 kinase. The protein also interacts with Cdk4 and inhibits the Cdk4/cyclin D1 enzyme. Surprisingly, dbpA(Cdelta) does not bind Cdk2 in the two-hybrid assay nor does it inhibit Cdk2 activated by cyclin A. It could be that dbpA's ability to inhibit Cdk5 and Cdk4 reflects an apparent cross-talk between distinct signal transduction pathways controlled by dbpA on the one hand and Cdk5 or Cdk4 on the other.     

Structural Basis for the Different Stability and Activity between the Cdk5 Complexes with p35 and p39 Activators

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.     

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.     

Structural and functional evidence that Nck interaction with CD3epsilon regulates T-cell receptor activity

Recruitment of signaling molecules to the cytoplasmic domains of the CD3 subunits of the T-cell receptor (TCR) is crucial for early T-cell activation. These transient associations either do or do not require tyrosine phosphorylation of CD3 immune tyrosine activation motifs (ITAMs). Here we show that the non-ITAM-requiring adaptor protein Nck forms a complex with an atypical PxxDY motif of the CD3ε tail, which encompasses Tyr166 within the ITAM and a TCR endocytosis signal. As suggested by the structure of the complex, we find that Nck binding inhibits phosphorylation of the CD3ε ITAM by Fyn and Lck kinases in vitro. Moreover, the CD3ε-Nck interaction downregulates TCR surface expression upon physiological stimulation in mouse primary lymph node cells. This indicates that Nck performs an important regulatory function in T lymphocytes by inhibiting ITAM phosphorylation and/or removing cell surface TCR via CD3ε interaction.     

The protein SET binds the neuronal Cdk5 activator p35nck5a and modulates Cdk5/p35nck5a activity.

The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35(nck5a) or its isoform, p39(nck5ai). To identify novel p35(nck5a)- and p39(nck5ai)-binding proteins, fragments of p35(nck5a) and p39(nck5ai) were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35(nck5a) and p39(nck5ai). Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35(nck5a) through its binding to p35(nck5a). The p35(nck5a)-interacting region was mapped to a predicted alpha-helix in SET. When cotransfected into COS-7 cells, SET and p35(nck5a) displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/p35(nck5a) from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35(nck5a), but not that of Cdk5/p25(nck5a), was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p35(nck5a).     

Identification of ribosomal protein L34 as a novel Cdk5 inhibitor

The cell cycle is regulated by sequential activation, inactivation of cyclin dependent kinases (Cdk-s). Like all other Cdk-s, the catalytic subunit of Cdk5 is present in cycling cells. However, its highest concentration is found in differentiated neurons, and the only known protein that activates Cdk5 (i.e., p35) is expressed solely in the brain. Active Cdk5 is thought to be involved in the in vivo phosphorylation of the neurofilament proteins and tau which are hyperphosphorylated in neurodegenerative diseases. Recent reports suggest that Cdk5 may also contribute to cellular differentiation. Therefore, it would not be unusual to surmise that there exist specific proteins that regulate Cdk5 activity in cycling cells. In order to find if this was true, a cDNA library prepared from HeLa cells was screened using the yeast-two-hybrid system. The 60S ribosomal protein, L34, was identified as a Cdk5-interacting protein. Biochemical analyses reveal that L34 cannot activate Cdk5 but potently inhibits the p35-activated kinase. L34 also interacts with Cdk4 and, in parallel, inhibits the Cdk4/cyclin D1 activity. Interestingly, L34 does not interact with Cdk2 in the two-hybrid assay nor does it inhibit the Cdk2/cyclin A enzyme. The fact that a ribosomal protein inhibits Cdk5 and Cdk4 may suggest that these two kinases have a cellular role in translational regulation.     

Cdk5--p39 is a labile complex with the similar substrate specificity to Cdk5--p35

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5--p39. In fact, kinase-active Cdk5--p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5--p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5--p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5--p39 complex showed similar substrate specificity to that of Cdk5--p35, but with opposite sensitivity to detergent. Cdk5--p39 was inactivated by Triton X-100, whereas Cdk5--p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5--p35 and Cdk5--p39 suggests their distinct roles under different regulation mechanisms in neurons.     

A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP-dependent protein kinase

While attempting to isolate a cDNA clone for the catalytic subunit of the bovine cAMP-dependent protein kinase, we have isolated cDNAs which code for a protein slightly different than the known amino acid sequence. The alternate cDNA was identified by screening a bovine pituitary cDNA library using synthetic oligonucleotides predicted from the known amino acid sequence of the catalytic subunit. The cDNA which we identified, encodes a protein which is 93% identical to the known amino acid sequence of the bovine catalytic subunit. It seems likely that this cDNA represents a previously undiscovered catalytic subunit of the cAMP-dependent protein kinase. The mRNA for the alternate catalytic subunit is different in size from the mRNA coding for the previously known catalytic subunit and also has a different tissue distribution. These findings suggest that there are at least two different genes for the catalytic subunit. The differences in amino acid sequence and tissue distribution suggest the possibility of important functional differences in the two enzymes.     

Molecular basis for the specificity of p27 toward cyclin-dependent kinases that regulate cell division

The cyclin-dependent kinase inhibitors (CKIs) bind to and directly regulate the catalytic activity of cyclin-dependent kinase (Cdk)/cyclin complexes involved in cell cycle control and do not regulate other, closely related Cdks. We showed previously that the CKI, p27, binds to Cdk2/cyclin A though a sequential mechanism that involves folding-on-binding. The first step in the kinetic mechanism is interaction of a small, highly dynamic domain of p27 (domain 1) with the cyclin subunit of the Cdk2/cyclin A complex, followed by much slower binding of a more lengthy and less flexible domain (domain 2) to Cdk2. The second step requires folding of domain 2 into the kinase inhibitory conformation. Rapid binding of p27 domain 1 to cyclin A tethers the inhibitor to the binary Cdk2/cyclin A complex, which reduces the entropic barrier associated with slow binding of domain 2 to the catalytic subunit. We show here that p27/cyclin interactions are an important determinant of p27 specificity towards cell cycle Cdks. We used surface plasmon resonance, limited proteolysis, mass spectrometry, and NMR spectroscopy to study the interaction of p27 with Cdk2/cyclin A, and with another Cdk complex, Cdk5/p25, that is involved in neurodegeneration. Importantly, Cdk5/p35 (the parent complex of Cdk5/p25) is not regulated by p27 in neurons. Our results show that p27 binds to Cdk5 and Cdk2 with similar, slow kinetics. However, p27 fails to interact with p25 within the Cdk5/p25 complex, which we believe prevents formation of a kinetically trapped, inhibited p27/Cdk5/p25 complex in vivo. The helical topology of p25 is very similar to that of cyclin A. However, p25 lacks the MRAIL sequence in one helix that, in the cell cycle cyclins, mediates specific interactions with domain 1 of p21 and p27. Our results strongly suggest that p21 and p27, related Cdk inhibitors, select their cell cycle regulatory Cdk targets by binding specifically to the cyclin subunit of these Cdk/cyclin complexes as a first step in a sequential, folding-on-binding mechanism.     

Nck in a complex containing the catalytic subunit of protein phosphatase 1 regulates eukaryotic initiation factor 2alpha signaling and cell survival to endoplasmic reticulum stress

Stress imposed on the endoplasmic reticulum (ER) induces the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51. This results in transient inhibition of general translation initiation while concomitantly activating a signaling pathway that promotes the expression of genes whose products improve ER function. Conversely, dephosphorylation of eIF2alphaSer51 is accomplished by protein phosphatase 1 (PP1c) complexes containing either the protein CReP or GADD34, which target PP1c to eIF2. Here, we demonstrate that the Src homology (SH) domain-containing adaptor Nck is a key component of a molecular complex that controls eIF2alpha phosphorylation and signaling in response to ER stress. We show that overexpression of Nck decreases basal and ER stress-induced eIF2alpha phosphorylation and the attendant induction of ATF4 and CHOP. In contrast, we demonstrate that the mouse embryonic fibroblasts lacking both isoforms of Nck (Nck1-/-Nck2-/-) show higher levels of eIF2alpha phosphorylation and premature induction of ATF4, CHOP, and GADD34 in response to ER stress and finally, are more resistant to cell death induced by prolonged ER stress conditions. We establish that a significant amount of Nck protein localizes at the ER and is in a complex with eIF2 subunits. Further analysis of this complex revealed that it also contains the Ser/Thr phosphatase PP1c, its regulatory subunit CReP, and dephosphorylates eIF2alpha on Ser51 in vitro. Overall, we demonstrate that Nck as a component of the CReP/PP1c holophosphatase complex contributes to maintain eIF2alpha in a hypophosphorylated state. In this manner, Nck modulates translation and eIF2alpha signaling in response to ER stress.     

Identification of novel substrates for Cdk5 and new targets for Cdk5 inhibitors using high-density protein microarrays

Cyclin-dependent kinase (Cdk) 5 is a serine/threonine kinase that plays an important role during CNS development and its dysregulation is causally involved in the process of neuronal degeneration. To date more than 20 Cdk5 substrates have been identified and the number of Cdk5 substrates is still increasing. The different cellular functions of Cdk5 and its substrates are not completely known at present. High-throughput protein microarray technology is a powerful tool to identify a large number of potential kinase substrates in parallel under the same experimental conditions. Using Protoarray protein microarrays we identified protein phosphatase 1, regulatory subunit 14A (PPP1R14A) as a novel substrate of Cdk5/p25. Phosphorylation was confirmed in two secondary assays. Our findings may contribute to the elucidation of the physiological function of Cdk5 in synaptic signalling. Functional Kinome Arrays were validated in a second set of experiments to characterize the selectivity of the Cdk5 inhibitor indolinone A. This lead to the identification of two additional kinases that are targeted by this compound and may provide a deeper understanding of its neuroprotective mode of action. However, several false negative results possibly due to a denatured or inactive conformation of the arrayed proteins, sound a note of caution when using protein array techniques.     

A Novel Member of the Cyclin-Dependent Kinase Family in Paramecium tetraurelia

Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The 'PSTAIRE' region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast p13suc1, even though it has better conservation of p13suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.