Interaction of cyclin-dependent kinase 5 (Cdk5) and neuronal Cdk5 activator in bovine brain.

Neuronal cdc2-like kinase (Nclk) purified from bovine brain is a heterodimer of Cdk5 and an essential 25-kDa regulatory subunit (Lew, J., and Wang, J. H.(1995) Trends Biochem. Sci. 20, 33-37). The regulatory subunit is an N-terminal truncated derivative of a 35-kDa protein expressed specifically in brain, hence the name neuronal Cdk5 activator, p25/p35. In this study, we probe the relationship between the two different forms of Nck5a and their interaction with and activation of Cdk5 in bovine brain extract. Using protein fractionation procedures in combination with Western blot analysis and protein kinase assay, three forms of Cdk5 have been detected in bovine brain: a monomeric Cdk5 that can be activated by bacterially expressed GST-p21, a heterodimer of Cdk5 and p25 that displays high kinase activity, and a Cdk5•p35 complex that is inactive and refractory to GST-p21 activation. Analysis of the Cdk5•p35 complex by gel filtration chromatography indicated that the complex was part of a macromolecular structure with a molecular mass of 670 kDa. When the macromolecular complex was subjected to gel filtration chromatography in the presence of 10% ethylene glycol, the fractions containing both p35 and Cdk5, although eluting at the same position as control, displayed high kinase activity. The result is compatible with the suggestion that the macromolecular complex contained a kinase inhibitory factor that dissociated from the complex in 10% ethylene glycol.

The animal cell division cycle is controlled by the coordinated actions of a family of protein Ser/Thr kinases, called cdc2-like kinases, which are activated and deactivated at discrete phases of the cell cycle. Members of the cdc2-like kinase family are heterodimers of a cdc2-related protein called cyclin-dependent kinase (Cdk) 1 and a cyclin. The activity of a cdc2-like kinase in dividing cells is usually dictated by the level of the cyclin subunit as well as the phosphorylation state of the Cdk subunit. In general, the catalytic activities of cell cycle cdc2-like kinases depend on the phosphorylation of a threonine residue (Thr-161 or its equivalent) by a specific CDK-activating kinase, whereas phosphorylation of a threonine or a tyrosine residue at the nucleotide binding loop of Cdk subunit by specific inhibitory kinases results in the inactivation of the kinases (for review, see Refs. [1][2][3][4][5][6][7][8][9][10]. In addition, cdc2-like kinases undergo functional and regulatory significant associations with a large number of cellular proteins, which include a family of low molecular weight Cdk inhibitory proteins (11)(12)(13)(14)(15)(16)(17), transcription factors, and Rb and Rb-related proteins (18 -28). In a number of reports, Cdks and cdc2-like kinases have been shown to exist within macromolecular complexes (26 -32). While it is logical to suggest that these macromolecular complexes represent the functional and regulatory significant association of Cdks and/or cdc2-like kinases with defined cellular proteins, systematic studies of such complexes have not been carried out.
Recently, a cdc2-like kinase has been discovered and purified from bovine brain as a proline-directed kinase (33,34) or a tau protein kinase II (35). This enzyme has been implicated in the regulation of neurocytoskeletal proteins, and a loss in the regulation of the enzyme has been suggested to be involved in Alzheimer pathology (34,36). The purified protein kinase is a heterodimer of a 33-kDa catalytic subunit and a 25-kDa regulatory subunit (33). Molecular cloning has identified the 33-kDa subunit as Cdk5 and the 25-kDa regulatory subunit as a proteolytic derivative of a novel neuron-specific 35-kDa protein (31,34,37). The regulatory subunit is essential for the kinase activity and only expresses in the central nervous system. Therefore, it has been designated as the neuronal Cdk5 activator, Nck5a (p25 nck5a /p35 nck5a ), and the heterodimeric holoenzyme as the neuronal cdc2-like kinase, Nclk (38 -40). Since neurons in adult brain do not divide, Nclk is unique in being a cdc2-like kinase not involved in cell division.
As a cdc2-like kinase, Nclk is expected to share many common regulatory and functional properties with other family members. Thus, the enzyme is composed of a catalytic subunit and a positive regulatory subunit. All the regulatory phosphorylation sites found in other Cdks are conserved or conservatively substituted (Ser to Thr) in Cdk5 (34,41). On the other hand, for its neuronal functions, Nclk may possess unique regulatory properties that distinguish itself from the cell cycle regulatory cdc2-like kinases. For example, while Nck5a displays a cyclin-like activity in that it activates the catalytic subunit, it shows little, or at best, marginal amino acid sequence homology to cyclins (37,41). In addition, the activation of Cdk5 by Nck5a is independent of the Cdk-activating kinase, whereas cyclin activation of Cdks in many cases have been found to depend on the Cdk-activating kinase (38). A comparison of the regulatory properties of Nclk with those of the cell cycle regulatory cdc2-like kinases may shed new light on both cell cycle regulation and the control of neuronal functions.
Although the purified bovine brain Nclk is composed of Cdk5 and p25 nck5a , the intact p35 nck5a is the predominant form of the protein in crude brain extract (37). The relationship between the two forms of Nck5a and their interaction with Cdk5 in the brain extract are not known. In this study, we have examined the states of Cdk5 in crude bovine brain extract using protein fractionation procedures in combination with kinase activity and immunological analysis. The results suggest that Cdk5 exists in at least three forms: a free Cdk5 monomer that is readily activated by a bacterially expressed p21 nck5a , a heterodimer of Cdk5/p25 nck5a that is highly active, and a Cdk5/ p35 nck5a form in association with a macromolecular complex that is neither enzymatically active nor activable by the bacterially expressed GST-p21 nck5a .

Protein Fractionation Procedures
Tissue Homogenate-All procedures were carried out at 4°C. 1 kg of fresh bovine brain was homogenized in a Waring blender in 1.5 liters of buffer A (25 mM Hepes, pH 7.2, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.6 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml aprotinin, 2 g/ml antipain, 0.3 mg/ml benzamidine, 0.1 mg/ml soybean trypsin inhibitor) for a 15-s burst on low speed and then two more times at 15 s on high speed. The crude homogenate was centrifuged at 12,000 ϫ g for 25 min in a Beckman JA-10 rotor, and the supernatant was further centrifuged at 120,000 ϫ g for 30 min in a Beckman Ti-45 rotor.
FPLC Mono-S Column Chromatography-40 mg of the 120,000 ϫ g supernatant was diluted 3-fold with buffer A and injected at 0.5 ml/min onto a Pharmacia Mono-S (HR 5/5) column for initial characterization of Nck5a and Cdk5 from bovine brain. The column was washed with 20 ml of buffer A and eluted with a 30-ml linear gradient (0 -0.5 M NaCl in buffer A) at 0.5 ml/min. 1-ml fractions were collected.
DEAE-Hydroxylapatite Column Chromatography-1.3 liters of the 120,000 ϫ g supernatant was loaded at 2 ml/min onto a 400-ml DEAE-Sepharose CL-6B column directly coupled to a 100-ml hydroxylapatite column pre-equilibrated with buffer A. After sample application, the DEAE-Sepharose CL-6B column was washed with 1 bed volume of buffer A. The DEAE-Sepharose CL-6B column was removed, and the hydroxylapatite column was washed with 500 ml of buffer A. The hydroxylapatite column was eluted with a linear gradient of K 2 HPO 4 (0 -400 mM) at 1 ml/min. 12-ml fractions were collected.
FPLC Superose-12 Gel Filtration Column Chromatography-Each of the pooled fractions (fractions I, II, and III) from the hydroxylapatite column were concentrated at 4°C by reverse dialysis against Aquacide II (Calbiochem). 3 ml of the concentrated sample was applied to an FPLC Pharmacia Superose-12 (HR 16/50) gel filtration column (100 ml) equilibrated in buffer A without 0.1 mg/ml soybean trypsin inhibitor and with 0.15 M NaCl at 0.5 ml/min. 1-ml fractions were collected. Molecular weight calibration of the Superose-12 column was performed under the same conditions using a gel filtration calibration kit from Bio-Rad (bovine thyroglobulin, 670 kDa; ferritin, 440 kDa; bovine ␥ globulin, 158 kDa; chicken ovalbumin, 44 kDa; horse myoglobin, 17 kDa; vitamin B-12, 1.35 kDa).
FPLC Superose-6 Gel Filtration Column Chromatography-The pooled fraction (fraction III) from the hydroxylapatite column was concentrated as described above. 3 ml of the concentrated sample was applied to an FPLC Superose-6 (XK 16/70) gel filtration column (120 ml) equilibrated in buffer A without 0.1 mg/ml soybean trypsin inhibitor and with 0.15 M NaCl at 0.5 ml/min. 1-ml fractions were collected. Molecular weight calibration of the Superose-6 column was performed as described above.

In Vitro Reconstitution Assays
Either bacterially expressed GST-p21 nck5a (0.1 g) or bacterially expressed GST-Cdk5 (0.1 g) was combined with 10 l of column fraction and incubated at room temperature for 40 min before histone H1 peptide phosphorylating activity was determined as described above.

Expression and Purification of GST-fusion Proteins from Bacteria
GST-fusion proteins were purified as described (43). pGEX-2T recombinant plasmid containing either Cdk5 or p21 nck5a , a truncated form of p25 nck5a (38), was transformed into Escherichia coli strain DH5␣. The host cells were cultured in 4 liters of LB medium containing 100 g/ml ampicillin to A 600 nm ϭ ϳ0.7 at 37°C. The expression of GST-fusion proteins was induced with 0.4 mM isopropyl-␤-D-thiogalactopyranoside (Life Technologies, Inc.) at room temperature overnight. The cells were harvested by centrifugation at 5,000 rpm for 15 min, washed with 1 ϫ phosphate-buffered saline, suspended in 30 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1 mM DTT, 0.25 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml antipain), and lysed three times using a French press (SLM Aminco, Silver Spring, MD) with the cell pressurized to 16,000 psi. The resulting lysate was centrifuged at 18,000 rpm for 20 min. The supernatant was incubated for 1 h at 4°C with 4 ml of glutathione-agarose (Sigma) previously equilibrated with the same buffer. After loading the slurry onto a 10-ml column, the column was washed with 10 bed volumes of 1 ϫ phosphate-buffered saline supplemented with 0.25 M KCl, 0.1% Tween 20, 1 mM DTT, 0.25 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml antipain and followed by 5 bed volumes of 1 ϫ phosphate-buffered saline supplemented with 1 mM DTT. The expressed GST-fusion proteins were eluted by 5 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, 1 mM DTT.

SDS-Polyacrylamide Gel Electrophoresis, Immunoblots, and Antibodies
SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (44) in 12.5% vertical slab gels. For Western blot analyses, ϳ30 g total protein was loaded onto each lane. After electrophoresis, proteins were transferred to Immobilon-P membrane (Millipore). The blots were probed with the indicated primary antibodies, followed by incubation with a 1/5000 dilution of the affinity-purified goat anti-rabbit IgG conjugated with alkaline phosphatase (Jackson Immunoresearch Laboratories Inc.) and detected with 10 ml of 100 mM Tris buffer, pH 9.5, containing 100 mM NaCl, 5 mM MgCl 2 , 0.37 mM p-nitro blue tetrazolium chloride, and 0.41 mM 5-bromo-4-chloro-3-indolyl phosphate disodium salt. Specific rabbit polyclonal antibodies were generated in our laboratory against bacterially expressed p25 nck5a and Cdk5 proteins, a C-terminal p25 nck5a peptide (Q 284 VFSDLKN-ESGQED 297 ), and an N-terminal p25 nck5a peptide (P 109 PASQLS-GSQTG 120 ). All were used at a concentration of 2 g/ml except the latter, which was used at 100 g/ml.
For the ECL system, the blot was probed with 1/2000 dilution of a rabbit polyclonal Cdk5 (C-8) antibody from Santa Cruz Biotechnology, Inc., followed by incubation with goat anti-rabbit IgG conjugated with horseradish peroxidase and developed as specified by the manufacturer (Amersham Corp.).

Protein Concentration Determination
Protein concentration was determined from A 280 nm values or by Bradford microassay (45).

RESULTS AND DISCUSSION
To analyze the relationship between p25 nck5a and p35 nck5a and their interactions with Cdk5 in brain extracts, we attempted the fractionation of the two forms of Nck5a in a 120,000 ϫ g fraction of the brain extract on various columns. Initial characterization using specific antibodies showed that both Cdk5 and Nck5a were quantitatively absorbed on a hydroxylapatite column or a cation exchanger such as an FPLC Mono S column but negligibly bound to anion exchangers such as DEAE-Sepharose and FPLC Mono Q columns. When a small amount of crude sample (40 mg) of fresh bovine brain 120,000 ϫ g extract was applied to an FPLC Mono S (HR 5/5) column, and the column eluents from a linear gradient of 0 -0.5 M NaCl were analyzed by Western blot analyses using Cdk5-and Nck5a-specific antibodies, Cdk5 was found to be eluted from the column over the entire salt gradient, whereas p25 nck5a and p35 nck5a were eluted separately, each within a narrow range of the salt gradient. The result was in agreement with a postulate that there were at least three forms of Cdk5 in the crude bovine brain sample: a p35 nck5a -complexed form, a p25 nck5a -complexed form, and a form not associated with Nck5a. To analyze the activity states of the different forms of Cdk5, the column eluents were assayed for histone H1 kinase activity in the presence and absence of GST-p21 nck5a , a bacterially expressed truncated form of p25 nck5a previously shown to activate the bacterially expressed Cdk5 (38). When the kinase assay was carried out in the absence of GST-p21 nck5a , a small amount of kinase activity was detected, exclusively in fractions containing p25 nck5a . In fractions containing Cdk5 but without p25 nck5a or p35 nck5a , high levels of the kinase activity could be demonstrated in the presence of GST-p21 nck5a . On the other hand, fractions containing Cdk5 and p35 nck5a appeared to have little or no activity in the presence and absence of GST-p21 nck5a . Thus, it may be further suggested that the three forms of Cdk5 have different histone H1 kinase activity characteristics. The Cdk5/p25 nck5a is the only active species of Cdk5, the uncomplexed Cdk5 is inactive but can be activated by Nck5a, and Cdk5/p35 nck5a is inactive as well as refractory to Nck5a activation.
To further test this suggestion, the 120,000 ϫ g bovine brain extract was analyzed by different column chromatographic procedures, a procedure combining both DEAE-Sepharose and hydroxylapatite column. In comparison with a Mono S chromatography, this procedure has the advantage that a large amount of the sample can be processed, and the separated fractions may be further analyzed. A sample (1,300 ml) of the 120,000 ϫ g fraction was loaded onto a 400-ml DEAE-Sepharose column directly coupled to a 100-ml hydroxylapatite column. After sample application, the columns were disconnected, and the hydroxylapatite column was eluted with a linear gradient of K 2 HPO 4 (0 -400 mM). The eluted proteins were analyzed for histone H1 peptide-phosphorylating activity in the presence and absence of GST-p21 nck5a , a bacterially expressed truncated form of p25 nck5a that is capable of activating a bacterially expressed Cdk5 (38), and immunoblotted with either a Nck5a-or Cdk5-specific antibodies. As shown in Fig. 1A, in the absence of GST-p21 nck5a , a small peak of endogenous kinase activity eluted at 280 mM K 2 HPO 4 . In the presence of GST-p21 nck5a , the kinase activity was markedly increased. The peak of the GST-p21 nck5a -activated kinase activity eluted slightly before that of the endogenous kinase activity (see Fig. 1A, inset). This indicates that the majority of Cdk5 exists in an inactive form but can be activated by GST-p21 nck5a .
Immunoblot analysis of the eluted proteins with a Nck5aspecific antibody showed two major immunoreactive bands with molecular weights corresponding to p25 nck5a and p35 nck5a (Fig. 1B). These two proteins were partially separated by hydroxylapatite column chromatography: the 25-kDa protein eluted between 250 and 330 mM K 2 HPO 4 , whereas the 35-kDa protein eluted between 280 and 400 mM K 2 HPO 4 . On the other hand, Cdk5 eluted between 140 and 400 mM K 2 HPO 4 (Fig. 1, C  and D). At K 2 HPO 4 Ͼ 250 mM, Cdk5 eluted with one or both forms of Nck5a; however, at [K 2 HPO 4 ] Ͻ 250 mM, Cdk5 eluted without Nck5a. Interestingly, only the fractions that contained p25 nck5a appeared to have significant endogenous kinase activity (Fig. 1, A and B). The peak of the GST-p21 nck5a -activated kinase activity, however, was observed in fractions containing Cdk5 but neither p25 nck5a nor p35 nck5a . At [K 2 HPO 4 ] Ͼ 330 mM, Cdk5 eluted only with p35 nck5a and showed no kinase activity in the presence and absence of GST-p21 nck5a (Fig. 1,  A-D). Together, these results support the existence of three distinct forms of Cdk5 in bovine brain extract: (i) a free form that is inactive but activable by Nck5a, (ii) an active p25 nck5a complexed form, and (iii) a p35 nck5a complexed form that is neither active nor activable by Nck5a.
To further test the existence of three distinct forms of Cdk5 in bovine brain extract, the hydroxylapatite fractions were pooled into three different groups; fraction I (nos. 28 -34), fraction II (nos. 36 -42), and fraction III (nos. 44 -54), which were enriched with free Cdk5, p25 nck5a /Cdk5, and p35 nck5a ⅐Cdk5 complex, respectively. Each of the fractions was concentrated and further analyzed by FPLC Superose-12 gel filtration chromatography. As shown in Fig. 2, A and C, the majority of Cdk5 in fraction I eluted at a volume corresponding to the molecular mass of ϳ30 kDa and became highly active when the kinase assay was carried out in the presence of GST-p21 nck5a . This indicates that Cdk5 exists mainly as a monomeric form. When fraction II was analyzed by Superose-12 gel filtration chromatography, a single peak of endogenous kinase activity containing both p25 nck5a and Cdk5 eluted at a volume corresponding to the molecular mass of ϳ60 kDa (Fig. 3). Addition of GST-p21 nck5a to the p25 nck5a /Cdk5-containing fractions did not increase the kinase activity, suggesting that Cdk5 and p25 nck5a exists as a complex. There was a peak of GST-p21 nck5a -activated kinase activity at an elution volume corresponding to the molecular weight of ϳ30 kDa. However, this is expected because the separation of the free and p25 nck5a complexed Cdk5 forms on the hydroxylapatite column was incomplete, and the fraction II sample contained free Cdk5 (see Fig. 1). Analysis of fraction III by Superose-12 gel filtration chromatography showed that most of p35 nck5a and Cdk5 of the sample co-eluted at the void volume of the column (Fig. 4), thus raising the possibility that these proteins were in a complex of over 400 kDa. There was little or no histone H1 kinase activity associated with these fractions in the presence and absence of GST-p21 nck5a . Occasionally, a small amount of free Cdk5, which possessed GST-p21 nck5a -activated kinase activity, was observed.
While the existence of GST-p21 nck5a -activable Cdk5 and the highly active p25 nck5a /Cdk5 form was expected, the presence of the inactive p35 nck5a /Cdk5 form was a somewhat surprising finding. One possible explanation for the lack of kinase activity in these fractions is that the two proteins, although co-eluted by the gel filtration column chromatography, did not form a complex. A number of observations, however, argued against this explanation. For example, the fractions containing both proteins could not be activated by GST-p21 nck5a , suggesting that Cdk5 did not exist as a free form. Similarly, the addition to these column fractions of a bacterially expressed GST-Cdk5, which could be readily activated by GST-p21 nck5a (see Figs. 1 and 5), did not result in active kinase, thus arguing against the presence of free p35 nck5a . Furthermore, the fraction III sample was analyzed by FPLC Superose-6 gel filtration chromatography, and p35 nck5a and Cdk5 again co-eluted at a volume corre- sponding to a molecular mass of ϳ670 kDa (Fig. 5). Although co-immunoprecipitation analysis with either Nck5a-specific or Cdk5-specific antibodies could provide a direct evidence for the existence of the p35 nck5a ⅐Cdk5 complex, we have been unable to produce a precipitating Cdk5 or Nck5a antibody. A Cdk5-specific antibody (C-8) from Santa Cruz Biotechnology was tried and found to precipitate the p25 nck5a ⅐Cdk5 complex, but the antibody did not immunoprecipitate Cdk5 from the fractions containing Cdk5 and p35 nck5a .
Another possibility that might account for the lack of kinase activity in fractions containing both p35 nck5a and Cdk5 is that the complex contains a protein(s) that inhibits the kinase. Recently, a number of inhibitory proteins for the family of cdc2-like kinases have been discovered: p21 CIP1 (also known as CAP20, SDI1, or WAF1) as a general inhibitor of Cdks (11)(12)(13)(14)(15), p16 INK4 as an inhibitor of cyclin D1-D3/Cdk4 (16), and p27 KIP1 as an inhibitor of cyclin E/Cdk2 (17). Some of these exist complexed with the holoenzyme form of the kinases to form inactive kinase complexes (14,29,41). Therefore, attempts were made to remove the putative inhibitor protein(s) from the complex using either high concentrations of NaCl or ethylene glycol. When fraction III was incubated with 1 M NaCl and then chromatographed on Superose-6 FPLC column in the presence of 1 M NaCl, the protein elution (data not shown) and enzyme activity profiles (see Fig. 6) were unaltered from the control sample (Fig. 5). However, when the sample was treated with 10% ethylene glycol and then subjected to gel filtration chromatography in the presence of 10% ethylene glycol, the fractions containing both p35 nck5a and Cdk5, although eluting at the same position as the untreated sample (Fig. 5), displayed high kinase activity (Fig. 6). The observation was compatible with a suggestion that a low molecular weight inhibitor had been removed from the complex. An alternative explanation that ethylene glycol incubation caused a conformational change of the kinase from an inactive to an active state is unlikely since incubation of the sample in 10% ethylene glycol without subjecting the gel filtration chromatography did not result in activation of the kinase (data not shown). Taken together, our results suggest a potential interaction between a low molecular weight inhibitor and the p35 nck5a ⅐Cdk5 complex to form the inactive high molecular weight complex.
In summary, we observed three distinct Cdk5 molecular species in bovine brain extract: (i) a monomeric Cdk5 that can be activated by Nck5a, (ii) a p25 nck5a ⅐Cdk5 form that is intrinsically active, and (iii) a p35 nck5a ⅐Cdk5 form that is inactive and refractory to Nck5a activation. Further analysis of the fractions containing both p35 nck5a and Cdk5 indicated the presence of an inactive p35 nck5a ⅐Cdk5 high molecular weight complex. A number of recent papers support the presence of the p35 nck5a ⅐Cdk5 high molecular weight complex: (i) the existence of inactive macromolecular complex-containing multiple p21 subunits in insect Sf9 cell lysate where p21 has been suggested to act as a Cdk assembly factor to promote the association of cyclin and Cdk subunits (30), (ii) the identification of Cdkactivating kinase complex as a component of TFIIH (26 -28), (iii) the identification of cyclin-dependent kinase binding protein (p15 cdk-BP ) as a part of a high molecular weight complex in starfish oocyte lysate (32), and (iv) the existence of additional Cdk5-regulatory subunits (p60, p62, and p180) aside from p25 nck5a and p35 nck5a in rat neuronal cells (31,40). 2 Although FIG. 5. Superose-6 gel filtration column chromatography of fraction III. A, protein fractionation of fraction III by a Superose-6 column was carried out as described under "Experimental Procedures." A, --, A 280 nm value; q--q, endogenous kinase activity; E--E, kinase activity in the presence of GST-p21 nck5a ; --, kinase activity in the presence of GST-Cdk5. B, immunoblot analysis of the eluted proteins with a Nck5a-specific antibody. C, immunoblot analysis of the eluted proteins with a Cdk5-specific antibody.
FIG. 6. Superose-6 gel filtration column chromatography of fraction III in the presence of 10% ethylene glycol. A, protein fractionation of fraction III by a Superose-6 column was carried out as described under "Experimental Procedures." A, --, A 280 nm value; q--q, endogenous kinase activity. Kinase activity values ( --) after Superose-6 gel filtration column chromatography of fraction III in the presence of 1 M NaCl are presented. B, immunoblot analysis of the eluted proteins with a Nck5a-specific antibody. C, immunoblot analysis of the eluted proteins with a Cdk5-specific antibody. the molecular basis for the lack of kinase activity of the macromolecular complex is not known, initial experiments are compatible with the suggestion that the complex contains kinase inhibitory factor(s). Tests are underway to identify a possible low molecular weight inhibitor.