Profiling of p5,a 24 Amino Acid Inhibitory Peptide Derived from the CDK5 Activator,p35 CDKR1 Against 70 Protein Kinases

Abstract

Cyclin-dependent kinase 5 (CDK5) is a multifunctional serine/threonine kinase that regulates a large number of neuronal processes essential for nervous system development and function with its activator p35 CDK5R1. Upon neuronal insults, p35 is proteolyzed and cleaved to p25 producing deregulation and hyperactivation of CDK5 (CDK5/p25), implicated in tau hyperphosphorylation, a pathology in some neurodegenerative diseases. A truncated, 24 amino acid peptide, p5, derived from p35 inhibits the deregulated CDK5 phosphotransferase activity and ameliorates Alzheimer’s disease (AD) phenotypes in AD model mice. In the present study, we have screened a diverse panel of 70 human protein kinases for their sensitivities to p5, and a subset of these to p35. At least 16 of the tested protein kinases exhibited IC50 values that were 250 μM or less, with CAMK4, ZAP70, SGK1, and PIM1 showing greater sensitivity to inhibition by p5 than CDK5/p35 and CDK5/p25. In contrast, the p5 peptide modestly activated LKB1 and GSK3β. A sub set of kinases screened against p35 showed that activity of CAMK4 in the absence of calcium and calmodulin was also markedly inhibited by p35. The Cyclin Y-dependent kinases PFTK1 (CDK14) and PCTK1 (CDK16) were activated by p35 at least 10-fold in the absence of Cyclin Y and by approximately 50% in its presence. These findings provide additional insights into the mechanisms of action for p5 and p35 in the regulation of protein phosphorylation in the nervous system.

Keywords

Alzheimer’s disease cyclin-dependent kinase 5 activator p35 CDK5R1 tau phosphorylation

1 INTRODUCTION

CDK5 is a multifunctional serine/threonine kinase that is commonly found in diverse mammalian tissues; its activators p35 (CDK5R1) and p39 (CDK5R2) are particularly expressed in the nervous system. CDK5/p35 phosphotransferase activity is tightly regulated, and this kinase targets many substrate proteins involved in neurite outgrowth, neuronal migration, cortical lamination, synaptic activity, and survival [1 –7]. It has also been implicated as a key player in learning and memory [8 –11].

Deregulated hyperactivity of CDK5 has been linked to neurodegeneration in such disorders as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, among others [12 –21]. We and other laboratories have proposed that deregulated CDK5 arises under stress-induced cleavage of p35 into p25, a 25 kDa cytosolic fragment that stably binds to and hyperactivates CDK5 to aberrantly phosphorylate neuronal proteins leading to neurodegeneration [13 , 22–26]. To determine which amino acid sequences of p25 were responsible for hyperactivation of CDK5, we systematically truncated p25 from both N- and C-terminal domains. Among the truncated peptides, some larger peptides were better activators than p35; however, some peptides smaller than 16 kDa inhibited CDK5 phosphotransferase activity [27, 28]. The smallest peptide p5 (24 amino acids) was the most effective inhibitor of CDK5 activity. Subsequently, we could show that p5 inhibited CDK5/p25 in vivo and could reduce or ameliorate AD phenotypes in three different mouse models without affecting CDK5/p35 activity, indicating that the peptide may be an excellent therapeutic candidate [26 , 29–33].

Recently, we identified the amino acid residues in p5 that were the most critical for its inhibitory activity toward CDK5 with either p25 or p35 [28]. During the course of this study, we were intrigued to observe that at higher concentrations than required for strong inhibition of CDK5/p25 and CDK5/p35, the MAP kinase extracellular regulated kinase-1 (ERK1) was also inhibited by p5, whereas glycogen synthase kinase 3 beta (GSK3β) was slightly activated by p5. These unexpected findings prompted the present study to further identify other protein kinases that may be sensitive to the actions of p5.

The profiling of kinase inhibitors is a critical and important step for drug development and their therapeutic applications. Screening of the actions of a promising compound across a broad collection of protein kinases is essential for understanding its biological and therapeutic implications. Careful analyses of hundreds of different protein kinases against hundreds of small molecular inhibitors on large on-line databases such as DrugKiNET (http://www.drugkinet.ca) and Kinase SARfari (https://www.ebi.ac.uk/chembl/sarfari/kinasesarfari) reveal that many candidate therapeutic compounds can have surprising off-targets, so it is highly desirable to determine the selectivity and potency of the inhibitor on multiple kinases from different classes. Obtaining any off-target reactivities, and in some cases identifying new targets, may lead to novel therapeutic applications of the inhibitor.

The neurodegenerative diseases are irreversible, progressive brain diseases that slowly induce neuronal loss, compromise memory and cognitive function, and eventually descend into death. Multiple pathways reflecting the complexities of systemic aging are triggered in neurodegenerative disorders and, undoubtedly, networks of cross-talking kinases are involved. To characterize the mechanistic role of p5 peptide in these networks, we have studied its effect in modulating the phosphotransferase activities of seventy diverse kinases under identical in vitro assay conditions. This study reveals that p5 not only inhibits CDK5 hyperactivation and deregulation, but also inhibits many other kinases with potencies equal to or greater than CDK5, and may even activate other kinases, some identified as relevant to neurodegeneration, and in some cases, cancer.

2 MATERIALS AND METHODS

2.1 Quality control and reagents

The various recombinant human protein kinases employed in the target profiling process along with p35 protein (GST-tagged full-length human CDK5P35, Cat. No. C34-30G-20) were sourced from SignalChem Pharmaceuticals, Inc. (Richmond, BC, Canada). Most of these were expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag and purified. Note that the inactive and active forms of the same protein kinases that were tested in this study represent different batches of these enzymes and the inactive forms may have been used at higher concentrations than the active forms to allow successful measurement of phosphotransferase activities in view of their lower specific enzyme activities. Quality control testing was routinely performed on each of the protein kinases to ensure compliance to acceptable standards. [γ–³³P]ATP was purchased from PerkinElmer. The inhibitory peptide p5 was synthesized at Kinexus to greater than 90% purity and a stock solution was made in 10% DMSO. The stock solution was then diluted to form an assay stock solution and this was used to profile against the selected 70 protein kinases. The sequence of the p5 peptide and its relation to the p35 sequences and cyclins sequences are provided in Table 4 for comparison. All other materials were of standard laboratory grade.

2.2 Protein kinase assays

The in vitro assay conditions for the protein kinases were optimized to yield acceptable enzymatic activities. In addition, the assays were optimized to give high signal-to-noise ratios. A list of all of the substrates used to assay the 70 different protein kinases tested is provided in Supplementary Table 1. A radioisotope assay format was used for profiling each kinase and was performed in duplicate at ambient temperature for 20–30 min in a final volume of 25 μl according to the following assay conditions: 5 μl of diluted active protein kinase (∼10–50 nM final protein concentration in the assay); 5 μl of assay solution of specific substrates for each kinase; 5 μl of kinase assay buffer (25 mM MOPs, pH 7.2, 12 mM β-glycerophosphate, 25 mM MgCl_2, 5 mM EGTA, 2 mM EDTA, and 0.25 mM dithiothreitol); 5 μl of p5 peptide (0.125 to 2.5 mM stock), or 10% DMSO; and 5 μl of [γ–³³P] ATP (250 μM stock solution, 0.8 μCi). For assays profiling for p35, conditions were the same as above, except p5 was replaced by 5 μl of p35 protein (80 ng/ μl stock).

Each assay was initiated by the addition of [γ–³³P] ATP. After the incubation period, the assay was terminated by spotting 10 μl of the reaction mixture onto a Multiscreen phosphocellulose P81 plate. The plate was washed 3 times for approximately 15 min each in a 1% phosphoric acid solution. The radioactivity on the P81 plate was counted in the presence of scintillation fluid in a Trilux scintillation counter. Blank controls were set up which included all the assay components except the addition of the appropriate substrate (replace with equal volume of assay dilution buffer). The corrected activity for each protein kinase was determined by subtracting the respective blank control value. The intra-assay variability was determined to be less than 10%. Supplementary Tables 2–4 provide the raw data from these protein kinase assays.

3 RESULTS

3.1 Screening the human kinome for p5 sensitivity

Profiling p5 against a wide spectrum of protein kinases that are representative of the broader human kinome was undertaken to evaluate the effects of p5 on their respective phosphotransferase activities. The enzymes were usually generated from full-length wild-type human genes that did not harbor activating mutations, but were activated by pre-phosphorylation or cofactors. However, the receptor-tyrosine kinases used lacked their extracellular domains.

The results of these in vit ro kinase assays are presented in order of increasing inhibition by the p5 peptide at 500 μM concentration in Table 1 (raw data are available in Supplementary Table 2). The intra-assay variability was determined to be less than 10% and the averages of duplicate measurements are shown (recorded as cpm of radioactive product generated in 20–30 min assays). Inhibition of kinase phosphotransferase activity by the p5 peptide yields a negative value, while activation of target activity is positive. We considered changes of 20% or greater to be significant.

At 500 μM concentration, the p5 peptide exhibited strong inhibition (greater than 50% compared to controls without p5) of 44 of 70 different protein kinases that were tested (Table 1). Of these, 16 kinases showed >80% inhibition of the phosphotransferase activity compared to the matched control, including ABL1, CAMK4, CDK5/p25, CDK5/p35, CDK6, CDK7, ERK2, MEK1, MET, PAK1, PCTK1 (CDK16), PFTK1 (CDK14), Pim1, PKAc-alpha, SGK1, and ZAP70. Six protein kinases, i.e., CLK1, DAPK1, EIF2AK2, HIPK1, TAOK1, and ULK1, displayed greater than 90% inhibition by the p5 peptide. One protein kinase, LKB1 (M025-alpha, STRAD-alpha), showed an increase in activity in the presence of the p5 peptide. Since the inhibitor peptide features multiple serine residues as well as basic amino acids, it is possible that apparent increases in enzyme activity may reflect direct phosphorylation of the p5 peptide by LKB1. This may also be true for PKAc-alpha and Pim1, since the first serine residue in the p5 peptides occurs within a sequence motif (RXXSV) that is strongly recognized for phosphorylation by these protein kinases [34].

3.2 Potencies of p5 inhibition of the most sensitive protein kinases

These surprising findings raise a number of questions that could have very significant consequences. For the most inhibited kinases, we initially sought to determine the significance of inhibition by the p5 peptide. Accordingly, we followed up with a more comprehensive determination of the IC50 values for kinase inhibition by the p5 peptide. We selected the top 21 protein kinases that showed the greatest degree of inhibition (greater than 80% ) by 500 μM p5 peptide and assayed these enzymes with various concentrations (25–500 μM) of the p5 peptide with the same standardized radioactive assay methodology used previously. We also re-tested GSK3β, since in our previous study [28] this kinase exhibited a 24% increase in phosphotransferase activity with 500 μM p5 peptide.

The profiling data for p5 peptide, when measured against 22 protein kinases, showed moderate to strong inhibition with 18 out of the 22 kinases tested (Supplementary Table 3). Two of the kinases tested (GSK3β and PKAc-alpha) exhibited activities, which were higher than the control substrate. Consequently, GSK3β, like LKB1 does appear to be weakly activated by the p5 peptide. The rest of the protein kinases tested experienced inhibitions ranging from 15% up to 97% at 500 μM with the average being 80% inhibition.

With the measurement of the inhibition of most of these protein kinases with multiple concentrations of the p5 peptide, IC50 determinations and other kinetic parameters could be calculated using the “best fit” values (Supplementary Figure 1). The IC50 values provide the best indication of how potent the p5 peptide was for inhibiting each kinase and the results are provided in Table 2. The lowest IC50 value determined was 6 μM for CaMK4. There were 13 other kinases that had relatively low IC50 values ranging from 23 μM to 208 μM, including in order of most potently inhibited ZAP70, SGK1, PIM1, CDK5/p25, EIF2AK2, PFTK1 (CDK14), MET, PCTK1 (CDK16), TAOK1, MEK1, DAPK1, ABL1, ERK2, and CLK1. The other kinases tested had either higher IC50 values than 208 μM or we were not able to determine their IC50 values. Since four kinases had IC50 values much lower than CDK5/p25 (74 μM), the principal target of the p5 peptide, these findings suggest that p5’s therapeutic effects in vivo may be mediated through its effects on multiple protein kinases.

3.3 p35 effects on p5-sensitive protein kinases

The ability of the p5 peptide to inhibit p25 or p35 activation of CDK5 can be readily explained as a case of competitive inhibition, since the p5 amino acid sequence is identical to portions of these proteins. We wondered whether inhibition of many other protein kinases by the p5 peptide might also arise from an interference of their activation by cyclins and other cofactors. We also asked if p35 might directly activate some of these p5-sensitive protein kinases. Accordingly, we tested the effect of recombinant human p35 on the phosphotransferase activities of several of these enzymes. As shown in Table 3, our preparation of p35 at the concentration of 15 μg/ml (0.4 μM) produced more than 600-fold increase in the CDK5 activity with its substrate histone H1. However, at the same concentration, p35 had little or no significant effect on the activities of the active forms of ABL1, CAMK4, CLK1, DAPK1, EIF2AK2, ERK2, MEK1, MET, PIM1, SGK1, TAOK1, and ZAP70. On other hand, Cyclin Y-stimulated PCTK1 and PFTK1 kinases were further activated by approximately 50% by p35, and in the absence of Cyclin Y, these kinases were stimulated 100% or greater to levels not too different from that achieved with Cyclin Y. Furthermore, the unphosphorylated, inactive form of ERK2 showed a 5.8-fold increase in phosphotransferase activity with p35, and there was also a modest 57% increase in the enzymatic activity of unactive SGK1. Conversely, in the absence of calcium and calmodulin, CAMK4 exhibited some phosphotransferase activity, and this was totally abolished by p35.

4 DISCUSSION

The findings reported here have profound implications as to the physiological roles of the p35 CDK5R1 and its p25 product, which are potent activators of CDK5 activity. A number of studies have demonstrated that p35 acts as a “targeting” regulator for CDK5; its N-terminal head domain bound to the cell membrane is known to interact with many synaptic and cytoskeletal proteins (some of which are CDK5 substrates) and its presence confers specificity. The N-terminal region also protects CDK5/p35 from inhibition by p5 compared to the slightly more sensitive CDK5/p25, which lacks this region [28, 32]. To our knowledge, p35 and p25 have not been reported to activate or inhibit other protein kinases in situ.

Broad protein kinase activity screening was undertaken to ascertain specificity of action of the p5 peptide on diverse kinases. The aim of the present investigation was to identify the extent and potency of the inhibitory peptide on the phosphotransferase activities of a wide range of protein kinases. It is evident from this study that the p5 peptide, at 500 μM concentrations in test-tube assays with expressed and purified kinases, inhibits a wide range of protein kinases besides CDK5/p35 and CDK5/p25. Strong inhibition (>50% inhibition compared to control) by p5 was noted for 44 kinases, and half of these were inhibited at 80–95% at this concentration of p5. Many of the p5-sensitive protein kinases were cyclin-dependent protein kinases (CDKs). The mechanism by which p5 may inhibit other CDKs likely involves competitive inhibition of the correct binding of the optimal cyclin partners for each member of the CDK family. As shown in Table 4, the p5 sequence corresponds to the most conserved portion of the cyclin family of proteins in what is known as the cyclin box. Although the p35 CDK5R1 amino acid sequence differ from the cyclins, it still features many amino acid residues that are shared with other cyclins. Since these CDKs were assayed at submicromolar levels with their cyclin partners, it is probable that the p5 peptide at a much higher concentrations effectively competed and with the association of cyclins with their respective CDKs.

In view of inhibition of CDKs by p5, we tested the ability of p35 itself to activate those CDKs that were most potently inhibited by the p5 peptide. This included PFTK1 (CDK14) and PCTK1 (CDK16), which are both activated by Cyclin Y. p35 was able to stimulate the phosphotransferase activities of these protein kinases towards the substrate myelin basic protein in the absence of Cyclin Y to levels that were comparable to those recorded in the presence of Cyclin Y, and produced a further 50% increase in activity even when Cyclin Y was included. Consequently, it appears that these particular CDKs might also be activated by p35 in vivo. Like p35, Cyclin Y is also a very distinctive cyclin in its primary structure (Supplementary Table 5 and Supplementary Figure 2). Although, Cyclin Y also possesses a cyclin box motif similar to that of other cyclins, as shown in Table 4, it is more similar to the other cyclins than it is to p35 in this region. Therefore, it is unclear why p35 can substitute with Cyclin Y for activation of PFTK1 and PCTK1, although it may explain why the p5 peptide is inhibitory toward these particular CDKs.

Our findings that many other protein kinases besides CDKs were also inhibited by the p5 peptide was quite unexpected. Among the most potently affected protein kinases were CAMK4, ZAP70, SGK1, PIM1, EIF2AK2, MET, TAOK1, MEK1, and DAPK1, which exhibited IC50 values that were comparable or much lower than estimated for CDK5, PFTK1, and PCTK1. CAMK4 exhibited the greatest sensitivity to the p5 peptide at an IC50 concentration of 6.0 μM, which has significant implications for neurodegenerative disorders, since this kinase plays a key role in lymphocytes and neurons. This calcium/calmodulin-dependent protein-serine/threonine kinase regulates transcription factors involved in inflammation and memory consolidation. Hence, its regulation and deregulation may participate in neurodegenerative disorders such as AD where inflammation and synaptic dysfunction are evident. For example, in the hippocampus, CAMK4 phosphorylation of CREB1, a transcription factor, is essential to long term potentiation and memory consolidation [35]. It is unclear, however, how inhibition of this kinase by p5 might rescue an Alzheimer phenotype in AD model mice.

As for those other kinases showing low IC50 values (PIM1, SGK1, and ZAP1), regulation and deregulation of these kinases (and other kinases in the study) may be involved in nervous system function and neurodegeneration. The results from our in vitro studies with recombinant protein kinases call for more extensive investigations into the role of these kinases in cells and tissues. For example, death-associated protein kinase (DAPK1), a pro-apoptotic, calcium/calmodulin-binding kinase acts early in the apoptotic pathways before the cell is committed to death [36]. Its participates in a wide variety of apoptotic signaling pathways including gamma-interferon induced programmed cell death [37]. Although CDK5 and DAPK1 both are distinct kinases, involved in different signal transduction pathways, in neuronal function and neurodegeneration. While both kinases are inhibited by p5, it is important to study the respective mechanisms of their p5-mediated inhibition in similar neurodegenerative disease models. Since other kinases are inhibited by p5 its essential to study their role in neurodegeneration. If, as shown for CDK5 and DAPK1, that other kinases are implicated in neurodegeneration, they too may serve as targets for p5 or other small molecule drugs that may also be therapeutic candidates.

Footnotes

ACKNOWLEDGMENTS

This work was supported by the intramural research program of the U.S. National Institute of Neurological Disease and Stroke, National Institutes of Health.

Authors’ disclosures available online ().

Appendix

References

Nikolic

, Dudek

, Kwon

, Ramos

, Tsai

(1996) The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes Dev 10, 816–825.

Ohshima

, Ward

, Huh

, Longenecker

, Veeranna , Pant

, Brady

, Martin

, Kulkarni

(1996) Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc Natl Acad Sci U S A 93, 11173–11178.

Grant

, Sharma

, Pant

(2001) Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism. Eur J Biochem 268, 1534–1546.

Gupta

, Tsai

(2003) Cyclin-dependent kinase 5 and neuronal migration in the neocortex. Neurosignals 12, 173–179.

Hammond

, Tsai

, Tan

(2004) Control of cortical neuron migration and layering: Cell and non cell-autonomous effects of p35. J Neurosci 24, 576–587.

Angelo

, Plattner

, Giese

(2006) Cyclin-dependent kinase 5 in synaptic plasticity, learning and memory. J Neurochem 99, 353–370.

Jessberger

, Gage

, Eisch

, Lagace

(2009) Making a neuron: Cdk5 in embryonic and adult neurogenesis. Trends Neurosci 32, 575–582.

Humbert

, Lanier

, Tsai

(2000) Synaptic localization of p39, a neuronal activator of cdk5. Neuroreport 11, 2213–2216.

, Sun

, Zhang

, Takahashi

, Ma

, Vinade

, Kulkarni

, Brady

, Pant

(2001) Regulation of NMDA receptors by cyclin-dependent kinase-5. Proc Natl Acad Sci U S A 98, 12742–12747.

10.

Morabito

, Sheng

, Tsai

(2004) Cyclin-dependent kinase 5 phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95 in neurons. J Neurosci 24, 865–876.

11.

Fischer

, Sananbenesi

, Pang

, Lu

, Tsai

(2005) Opposing roles of transient and prolonged expression of p25 in synaptic plasticity and hippocampus-dependent memory. Neuron 48, 825–838.

12.

Patrick

, Zukerberg

, Nikolic

, de la Monte

, Dikkes

, Tsai

(1999) Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402, 615–622.

13.

Lee

, Kwon

, Li

, Peng

, Friedlander

, Tsai

(2000) Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 405, 360–364.

14.

Patzke

, Tsai

(2002) Cdk5 sinks into ALS. Trends Neurosci 25, 8–10.

15.

Cruz

, Tseng

, Goldman

, Shih

, Tsai

(2003) Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles. Neuron 40, 471–483.

16.

Cheung

, Ip

(2004) Cdk5: Mediator of neuronal death and survival. Neurosci Lett 361, 47–51.

17.

Kesavapany

, Li

, Amin

, Zheng

, Grant

, Pant

(2004) Neuronal cyclin-dependent kinase 5: Role in nervous system function and its specific inhibition by the Cdk5 inhibitory peptide. Biochim Biophys Acta 1697, 143–153.

18.

Giese

, Ris

, Plattner

(2005) Is there a role of the cyclin-dependent kinase 5 activator p25 in Alzheimer’s disease? Neuroreport 16, 1725–1730.

19.

Luo

, Vacher

, Davies

, Rubinsztein

(2005) Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: Implications for mutant huntingtin toxicity. J Cell Biol 169, 647–656.

20.

Guo

(2006) When good Cdk5 turns bad. Sci Aging Knowledge Environ 2006, pe5.

21.

Lopes

, Oliveira

, Agostinho

(2009) Cell cycle re-entry in Alzheimer’s disease: A major neuropathological characteristic? Curr Alzheimer Res 6, 205–212.

22.

Taniguchi

, Fujita

, Hayashi

, Kakita

, Takahashi

, Murayama

, Saido

, Hisanaga

, Iwatsubo

, Hasegawa

(2001) Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains. FEBS Lett 489, 46–50.

23.

Patzke

, Tsai

(2002) Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29. J Biol Chem 277, 8054–8060.

24.

Smith

, Mount

, Shree

, Callaghan

, Slack

, Anisman

, Vincent

, Wang

, Mao

, Park

(2006) Calpain-regulated p35/cdk5 plays a central role in dopaminergic neuron death through modulation of the transcription factor myocyte enhancer factor 2. J Neurosci 26, 440–447.

25.

Shukla

, Skuntz

, Pant

(2012) Deregulated Cdk5 activity is involved in inducing Alzheimer’s disease. Arch Med Res 43, 655–662.

26.

Shukla

, Zheng

, Mishra

, Amin

, Steiner

, Grant

, Kesavapany

, Pant

(2013) A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer’s disease phenotypes in model mice. FASEB J 27, 174–186.

27.

Amin

, Albers

, Pant

(2002) Cyclin-dependent kinase 5 (cdk5) activation requires interaction with three domains of p35. J Neurosci Res 67, 354–362.

28.

Binukumar

, Shukla

, Amin

, Bhaskar

, Skuntz

, Steiner

, Winkler

, Pelech

, Pant

(2015) Analysis of the Inhibitory Elements in the p5 Peptide Fragment of the CDK5 Activator, p35, CDKR1 Protein. J Alzheimers Dis 48, 1009–1017.

29.

Zheng

, Li

, Amin

, Albers

, Pant

(2002) A peptide derived from cyclin-dependent kinase activator (p35) specifically inhibits Cdk5 activity and phosphorylation of tau protein in transfected cells. Eur J Biochem 269, 4427–4434.

30.

Zheng

, Kesavapany

, Gravell

, Hamilton

, Schubert

, Amin

, Albers

, Grant

, Pant

(2005) A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons. EMBO J 24, 209–220.

31.

Kesavapany

, Zheng

, Amin

, Pant

(2007) Peptides derived from Cdk5 activator p35, specifically inhibit deregulated activity of Cdk5. Biotechnol J 2, 978–987.

32.

Zheng

, Amin

, Hu

, Rudrabhatla

, Shukla

, Kanungo

, Kesavapany

, Grant

, Albers

, Pant

(2010) A 24-residue peptide (p5), derived from p35, the Cdk5 neuronal activator, specifically inhibits Cdk5-p25 hyperactivity and tau hyperphosphorylation. J Biol Chem 285, 34202–34212.

33.

Sundaram

, Poore

, Sulaimee

, Pareek

, Asad

, Rajkumar

, Cheong

, Wenk

, Dawe

, Chuang

, Pant

, Kesavapany

(2013) Specific inhibition of p25/Cdk5 activity by the Cdk5 inhibitory peptide reduces neurodegeneration in vivo. J Neurosci 33, 334–343.

34.

Palaty

, Clark-Lewis

, Leung

, Pelech

(1997) Phosphorylation site substrate specificity determinants for the Pim-1 protooncogene-encoded protein kinase. Biochem Cell Biol 75, 153–162.

35.

Kang

, Sun

, Atkins

, Soderling

, Wilson

, Tonegawa

(2001) An important role of neural activity-dependent CaMKIV signaling in the consolidation of long-term memory. Cell 106, 771–783.

36.

Bialik

, Kimchi

(2006) The death-associated protein kinases: Structure, function, and beyond. Annu Rev Biochem 75, 189–210.

37.

Shohat

, Spivak-Kroizman

, Cohen

, Bialik

, Shani

, Berrisi

, Eisenstein

, Kimchi

(2001) The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism. J Biol Chem 276, 47460–47467.