(Received for publication, November 20, 1995)
From the
A synthetic peptide corresponding in sequence to residues
6-20 of p34, cdc2(6-20), and a
substitution analogue, cdc2(6-20)F15K19, which contains Thr-14 as
the only phosphorylation target were used as substrates to identify a
novel protein kinase in bovine thymus cytosol. The kinase catalyzed the
phosphorylation of Thr-14 in both peptides and was purified extensively
on the basis of its peptide phosphorylation activity. Upon
SDS-polyacrylamide gel electrophoresis analyses, the purified samples
consistently displayed a prominent 43-kDa protein band which could
undergo in gel autophosphorylation, thus suggesting that this
band represented the kinase protein. The suggestion was supported
further by the observation that both cdc2(6-20) peptide
phosphorylation and the autophosphorylation reaction of the 43-kDa
protein were inhibited by millimolar concentrations of cAMP. The kinase
was found to inactivate Cdc2/cyclin B, Cdk2/cyclin A, and neuronal
Cdc2-like kinase (Nclk), a heterodimer of Cdk5 and neuronal Cdk5
activator (Nck5a), under phosphorylation conditions. The
phosphorylation of Nclk by the purified thymus kinase occurred on Cdk5.
The monomeric form of Cdk5 was also phosphorylated by the kinase.
Phosphoamino acid and phosphopeptide analysis of the phosphorylated
Nclk revealed that Thr-14 of Cdk5 was the sole site of protein
phosphorylation. The results suggest that this thymus kinase is a novel
Cdk inhibitory protein kinase, distinct from the recently cloned dual
functional and membrane-associated Cdc2 inhibitory kinase, Myt1
(Mueller, P. R., Coleman, T. R., Kumagai, A., and Durphy, W. G.(1995) Science 270, 86-90).
Animal cells contain a family of Cdc2 homologous proteins which
are involved in the regulation of cell cycle
progression(1, 2, 3, 4, 5) .
These proteins, whose activities depend on their association with
cyclins, are called cyclin-dependent kinases, Cdks(6) . In
addition to depending on cyclins for activity, Cdks are regulated by
protein phosphorylation mechanisms involving a network of specific
protein kinases and protein phosphatases. For example, Cdc2 kinase is
negatively regulated during S and G phases by
phosphorylation on a specific tyrosine, Tyr-15, and a threonine, Thr-14
residue(7, 8, 9, 10) . Since these
residues are located in the nucleotide binding loop, one of the most
conserved regions of Cdc2 family proteins, these phosphorylations may
represent a general regulatory mechanism for Cdc2-like kinases. The
kinase catalyzing the phosphorylation of Tyr-15 of Cdc2 has been
identified as related to the protein product of the yeast cell cycle
regulatory gene wee1(11, 12, 13) .
On the other hand, the kinase that catalyzes the phosphorylation of
Cdc2 at Thr-14 has not been identified positively. A membrane fraction
purified from Xenopus eggs and HeLa cells was shown to promote
the phosphorylation of Cdc2 on both Tyr-15 and
Thr-14(14, 15) . The protein kinase responsible for
such a dual specific phosphorylation event has been cloned
recently(16) . The protein kinase, Myt1, contains a putative
transmembrane segment and is located exclusively in a membrane fraction
of cells. On the other hand, no Thr-14 kinase activity has been
reported in a cytosolic fraction of cells where a major population of
Cdc2 is located(17, 18) .
Not all the Cdks are cell cycle regulators. Neuronal Cdc2-like kinase (Nclk), a heterodimer of Cdk5 and a regulatory protein which is expressed specifically in neurons of the central nervous system, is a prime example of a Cdc2-like kinase not involved in cell cycle control (19, 20) . As a functionally unique Cdc2-like kinase, Nclk also displays unique molecular and regulatory properties that distinguish it from the cell cycle regulatory Cdc2-like kinases(20) . The regulatory subunit of the enzyme, neuronal Cdk5 activator (Nck5a), performs a cyclin-like function but shows only marginal amino acid sequence similarity to cyclins(19, 20, 21) . While most well characterized Cdks depend on an activating kinase, Cdk activating kinase(22, 23, 24) , in addition to cyclin, for kinase activity, the activation of Cdk5 by neuronal Cdk5 activator is independent of Cdk activating kinase(20, 25) .
The present study demonstrates the existence of a protein kinase in bovine thymus cytosol that induces the inactivation of Cdc2/cyclin B kinase, Cdk2/cyclin A kinase, and Nclk. Characterization of the kinase-catalyzed phosphorylation of Nclk indicates that the kinase, designated the Cdk T14 kinase, phosphorylates Cdk5 at the Thr-14 residue. The results suggest that this kinase may be the Cdk inhibitory protein Ser/Thr kinase that has been proposed to play cell cycle regulatory roles. In addition, the present study suggests that Nclk, like cell cycle regulatory Cdc2-like kinases, may be regulated by the inhibitory protein Ser/Thr phosphorylation mechanism.
For the Cdk inactivation assay, the reaction mixture containing
kinase buffer, 0.1 mM [-
P]ATP,
reconstituted Cdk2/pA-His-cyclin A, Nclk, or Cdc2/cyclin B purified
from sea star oocyte and the appropriate amount of the Cdk T14 kinase
was incubated for an appropriate time period (described in the figure
legends) at 30 °C. The Cdk kinase assay was started by addition of
the histone H1(9-18) peptide (90 µM). Final reaction
volume was 30 µl (50 µl in the case for Cdc2 due to low kinase
concentration of the original sample of Cdc2 kinase). The control
reaction was carried out under the identical conditions except that the
T14 kinase was not added.
Figure 1:
Demonstration of the cdc2 N-terminal
peptide kinase activities in bovine thymus. A, the 100,000
g cytosolic fraction prepared from 500 g of bovine
thymus was chromatographed on a 400-ml DEAE-Sepharose CL-6B column as
described under ``Materials and Methods.'' The 15-µl
aliquots of column fractions were assayed for cdc2(6-20) (
)
and cdc2(6-20)F15K19 (
) phosphorylation activity. -
- -, NaCl gradient. B, pooled
cdc2(6-20)F15K19 peptide kinase fraction of DEAE-Sepharose CL-6B
column was applied to an 80-ml hydroxylapatite column. After extensive
wash with buffer A, the column was eluted with 0-0.2 M/800-ml linear potassium phosphate gradient. The 12-µl
aliquots of column fractions were assayed for cdc2(6-20)
(
), cdc2(6-20)F15K19 (circo]), or cdc2(6-20)A14
(
) phosphorylation activity. - - -, potassium
phosphate gradient.
To test such a suggestion, fractions from the DEAE-Sepharose column containing the overlapping kinase activities were pooled and further analyzed. The pooled sample was fractionated on a hydroxylapatite column, and column fractions were analyzed for kinase activities using three different peptides: cdc2(6-20), cdc2(6-20)F15K19, and cdc2(6-20)A14, a substitution peptide with Thr-14 replaced by alanine. Fig. 1B shows that the pooled DEAE-Sepharose sample gave rise to two peaks of cdc2(6-20) peptide kinase activity on the hydroxylapatite column, and that only the second peak contained cdc2(6-20)F15K19 phosphorylation activity. On the other hand, only the first peak was capable of phosphorylating cdc2(6-20)A14. The observation that the first activity peak did not phosphorylate cdc2(6-20)F15K19 suggests that the kinase in the fractions was a tyrosine-specific rather than a dual specificity protein kinase. Similarly, the failure of the kinase of the second hydroxylapatite peak to phosphorylate cdc2(6-20)A14 suggested that the second peak contained a Thr-specific rather than a dual specificity kinase. An analogous peptide derived from the amino acid sequence residues 6 to 20 of Cdk5, cdk5(6-20) (KLEKIGEGTYGTVFK), was also tested and found to be an efficient substrate for the kinases of both peaks (not shown). The second hydroxylapatite kinase activity peak may represent the putative Cdk inhibitory protein Ser/Thr kinase since all peptides containing a threonine residue corresponding to Thr-14 could serve as efficient substrates, whereas the cdc2(6-20) peptide analogue with Thr-14 substituted by alanine was not a substrate. This kinase is designated as the Cdk T14 kinase in this paper.
As there are hundreds of protein Ser/Thr kinases in a
typical tissue or cell extract, the possibility that
cdc2(6-20)F15K19 peptide was phosphorylated by a kinase unrelated
to the Cdk T14 kinase had to be examined critically. One criterion for
establishing the identity of Thr-14 kinase is that the kinase has to
display the ability to inhibit active Cdk kinases. To address this
question, a sample of 100,000 g fraction from 500 g of
bovine thymus was processed through successive column chromatography
steps including DEAE-Sepharose, hydroxylapatite, and phenyl-Sepharose
columns as described in the legend of Fig. 2. Fractions
containing cdc2(6-20)F15K19 peptide kinase activity from the
phenyl-Sepharose column were pooled and applied to a FPLC Mono Q
column. Column fractions from the Mono Q column were then analyzed for
both the peptide kinase activity and the ability to inhibit a
reconstituted Cdk2/protein A-poly(His)-cyclin A (pA-His-cyclin A)
kinase. Fig. 2A shows that the Mono Q chromatography
profile of the peptide kinase activity correlated closely with the
Cdk2/pA-His-cyclin A kinase inhibitory activity, suggesting that the
two activities were derived from the same kinase.
Figure 2:
Inactivation of Cdk kinases by the Cdk T14
kinase. A, cytosolic fraction from 500 g of bovine thymus was
processed through DEAE-Sepharose and hydroxylapatite column
chromatography as shown in Fig. 1. The cdc2(6-20)F15K19
kinase activity emerged from the hydroxylapatite column was pooled and
applied onto a 10-ml phenyl-Sepharose CL-4B column pre-equilibrated
with buffer A. The column was washed with the same buffer, and the Thr
kinase was eluted with 0-65%/160-ml linear ethylene glycol
gradient. Post-phenyl-Sepharose kinase sample was subjected to FPLC
Mono Q chromatography. The 6-µl aliquots of column fractions were
assayed for cdc2(6-20)F15K19 kinase activity (). For the
Cdk2/pA-His-cyclin A kinase inactivation assay (
), the
6.5-µl aliquots of fractions were incubated with 23 units (1 unit
= 1 pmol of phosphate transferred/min) of Cdk2/pA-His-cyclin A
in 30 µl under phosphorylation conditions for 40 min, followed by a
30-min histone H1 peptide kinase assay. - - -, NaCl
gradient. B, upper panel, 1.5 units of sea star
Cdc2/cyclin B kinase was preincubated with 15 units of partially
purified Cdk T14 kinase (
) or control, a Mono Q fraction without
T14 kinase activity (
) under phosphorylation conditions for
various time periods indicated, and then assayed (30 min) for kinase
activity using histone H1(9-18) peptide. Lower panel,
Cdc2/cyclin B (1.5 units) was preincubated with various amounts of the
Cdk T14 kinase for 60 min, followed by a 30-min histone H1 peptide
phosphorylation assay. C, upper panel, 23 units of
reconstituted Nclk was preincubated with 48 units of the Cdk T14 kinase
for various time periods indicated, followed by 10-min histone H1
peptide phosphorylation assay. Lower panel, reconstituted Nclk
(23 units) was preincubated with various amounts of T14 kinase for 60
min, followed by a 10-min histone H1 peptide phosphorylation
assay.
In addition, the inhibitory activity of the Cdk T14 kinase was tested on two other members of the Cdc2-like kinase family: sea star oocytes Cdc2/cyclin B kinase (from Upstate Biological Inc.) and a reconstituted neuronal Cdc2-like kinase (Nclk) comprised of GST-Cdk5 and neuronal GST-Nck5a (neuronal Cdk5 activator). Fig. 2, B and C, shows the time courses and the dose dependence of the inhibition of Cdc2/cyclin B and Nclk by the Cdk T14 kinase, respectively. Almost complete inhibition could be achieved by using high concentrations of the Cdk T14 kinase or by a moderate concentration of the kinase with a long incubation time. These results, therefore, further support the suggestion that the cdc2(6-20)F15K19 kinase activity represents the putative protein Ser/Thr kinase that inhibits Cdk kinases by phosphorylating the Thr-14 residue in Cdks.
The enzyme sample emerged from the second FPLC Mono Q column; the last step of purification was highly purified but not yet homogeneous. SDS-PAGE analysis of the column fractions consistently revealed a protein band of apparent molecular mass of 43 kDa which represented the major protein component of the kinase fractions. Fig. 3A shows the protein elution profile and the peptide kinase activity profile as well as Nclk inhibitory activity of a typical Mono Q column chromatography. As same as the result in Fig. 2A, both activities comigrated perfectly. An SDS-PAGE analysis of the protein patterns of the column fractions as revealed by silver stain is shown in Fig. 3B. Note the presence of the prominent 43-kDa protein band (indicated by an asterisk) whose staining intensity correlated with the peptide kinase activity and Nclk inhibitory activity.
Figure 3:
Purification of the Cdk T14 kinase. A, post-Superose 12 sample (see ``Materials and
Methods'') of the Cdk T14 kinase was rechromatographed on a FPLC
Mono Q column. , cdc2(6-20)F15K19 phosphorylation by 6
µl of column fraction.
, inactivation of reconstituted Nclk
(8 units) by 7.5 µl of column fraction under the condition
described in Fig. 2A except that the reaction time of
Nclk was 20 min.
, protein profile;
- - -, NaCl gradient. B, Mono Q column
fractions of A (13.5 µl) were analyzed by 10%
SDS-PAGE/silver staining. The location of the major protein band of 43
kDa is marked by *.
To determine the protein concentration of the
purified kinase, a sample was subjected to SDS-PAGE, and the gel was
stained by Coomassie Brilliant Blue and then analyzed by densitometric
measurement. The intensity of the densitometric tracing of the sample
was then compared to a protein concentration calibration curve using
bovine serum albumin. Using the protein concentration so determined and
the kinase activity determined at 0.1 mM ATP and 0.5 mM cdc2(6-20)F15K19, the specific activity of the purified
kinase was determined as 0.12 µmol/min/mg (Table 1). In
addition, the densitometric analysis of the purified sample indicated
that 50% of total protein of the sample was represented by the 43-kDa
protein. The specific activity was, therefore, calculated as 0.24
µmol/min/mg if only the 43 kDa was considered. The enzyme was also
characterized in terms of its K and V
values using the peptide substrate. At the ATP
concentration of 0.1 mM, the K
of the
peptide and V
of the enzyme were found to be
0.58 mM and 0.37 µmol/min/mg, respectively. The K
for ATP was determined at 0.5 mM cdc2(6-20)F15K19 to be 80 µM.
Figure 4: In gel autophosphorylation of the Cdk T14 kinase. Proteins in the purified Cdk T14 kinase sample (48 µl) of the Cdk T14 kinase were separated by 10% SDS-PAGE and subjected to an in gel phosphorylation reaction (see ``Materials and Methods''). Left, Coomassie staining of the purified Cdk T14 kinase. Right, autoradiogram.
Although the experimental result of Fig. 4revealed the 43-kDa protein as a protein kinase, it did not necessarily indicate that the enzyme was the kinase responsible for the phosphorylation of cdc2(6-20)F15K19 peptide. During the study of regulatory properties of the purified thymus kinase, we found that relatively high concentrations (millimolar concentrations) of cAMP could cause marked inhibition of the kinase-catalyzed phosphorylation of cdc2(6-20)F15K19 peptide. The other nucleotides tested, including AMP, cGMP, and GMP, showed much weaker inhibitory activity toward the peptide phosphorylation activity. A dose-dependent inhibition of the kinase activity by cAMP as well as those by the other nucleotides are shown in Fig. 5A. The inhibition of the peptide phosphorylation activity occurs at nonphysiological concentrations of cAMP, suggesting that the inhibition is not a physiologically relevant phenomenon. However, we have used this cAMP effect to test whether or not the 43-kDa protein is the kinase responsible for the phosphorylation of cdc2(6-20)F15K19 peptide. Thus, a purified sample of the kinase was subjected to SDS-PAGE, and the effects of the various nucleotides on the in gel autophosphorylation of the 43-kDa protein were examined. Fig. 5B shows that the autophosphorylation of the 43-kDa protein, like the kinase activity toward cdc2(6-20)F15K19 peptide, was markedly inhibited by cAMP and weakly inhibited by AMP, cGMP, or GMP. The observation strongly suggests that the 43-kDa protein is the protein kinase catalyzing cdc2(6-20) peptide phosphorylation.
Figure 5:
Inhibition of the Cdk T14 kinase activity
by cAMP. A, the purified Cdk T14 kinase activity was assayed
for cdc2(6-20)F15K19 in the presence of various concentrations of
nucleotide. , GMP;
, cGMP;
, AMP;
, cAMP. B, the 43-kDa protein in the purified Cdk T14 kinase sample
was visualized by in gel autophosphorylation/autoradiography. In gel autophosphorylation reaction was carried out in the
presence of nucleotide. Lane 1, control; lane 2,
cGMP; lane 3, AMP; lane 4,
cAMP.
Figure 6:
Analysis of GST-Cdk5 phosphorylation by
the Cdk T14 kinase. GST-Cdk5, reconstituted Nclk, or GST-Nck5a was
subjected to phosphorylation reactions in the presence of
[-
P]ATP with or without the Cdk T14 kinase
and then isolated by using GSH-agarose. The isolated protein was
applied to 7.5% SDS-PAGE and analyzed by Western blot with
-Cdk5
antibody (left) and autoradiography (right). Lane
1, the Cdk T14 kinase + GST-Cdk5; lane 2, GST-Cdk5
alone; lane 3, the Cdk T14 kinase + GST-Nck5a; lane
4, reconstituted Nclk alone; lane 5, the Cdk T14 kinase
+ reconstituted Nclk.
To determine the amino acid residue phosphorylated in the peptide and protein substrates, the Cdk T14 kinase-phosphorylated GST-Cdk5 and the peptide substrates cdc2(6-20) and cdk5(6-20) were subjected to phosphoamino acid analysis. As shown in Fig. 7A, a-c, all the substrate tested contained phosphothreonine as the only phosphoamino acid. Addition of sodium orthovanadate which would inhibit protein-tyrosine phosphatases that might have contaminated the kinase sample used in the phosphorylation reaction had no effect (Fig. 6A, d). The same result was obtained for Cdk T14 kinase-phosphorylated Cdk2 protein (not shown).
Figure 7:
Determination of phosphorylation site of
Cdk5. A, phosphoamino acid analysis of Cdk T14
kinase-phosphorylated GST-Cdk5 and peptides. a, Cdk T14
kinase-phosphorylated Cdk5 protein; b, cdc2(6-20); c, cdk5(6-20); d, cdk5(6-20) + 50
µM NaVO
. B,
two-dimensional phosphotryptic peptide map of Cdk5 protein. The tryptic
peptide prepared from Cdk T14 kinase-phosphorylated GST-Cdk5 or
cdk5(6-20) was standardized by its radioactivity. The mixture of
them was then subjected to TLE/TLC.
To further identify the site of phosphorylation of GST-Cdk5 by the putative T14 kinase, the phosphorylated Nclk was subjected to tryptic digestion, and the resulting peptide mixture was analyzed by two-dimensional electrophoresis/chromatography and autoradiography. The phosphopeptide map was then compared to that of the control; the peptide cdk5(6-20) was phosphorylated by the purified kinase and then treated with trypsin. Both the control peptide and the tryptic peptide mixture displayed a single intense spot on the phosphopeptide map (not shown). The phosphopeptide of the sample appeared to have electrophoretic and chromatographic properties identical with the phosphopeptide map of a mixture of the control and the sample (the same amount of radioactivity) gave rise to a single spot (Fig. 7B). Together, these results indicate that Cdk5 was phosphorylated by the Cdk T14 kinase specifically on Thr-14.
In the present work, we have identified in bovine thymus cytosol a novel protein kinase that is capable of in vitro inactivation of a number of Cdc2-like kinases, including Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (Nclk). On the basis of the enzyme specificity toward peptide substrates and chemical characterizations of the enzyme-catalyzed Nclk phosphorylation reaction, the novel kinase is suggested to be specific for the negative regulatory threonine residue of Cdks, i.e. Thr-14, hence the name Cdk T14 kinase. The cytosolic localization and strict specificity toward Thr-14 clearly distinguish the Cdk T14 kinase identified in this study from the reported dual specificity and membrane-associated kinase (14, 15, 16) . Since most of the Cdks exist in cell cytosol(17, 18) , it is tempting to suggest that the Cdk T14 kinase is the major enzyme responsible for the inhibitory phosphorylation of Cdks on Thr-14. However, additional characterizations of the enzyme, particularly in terms of its activity and regulation in relation to cell cycle progression, have to be carried out to test such a suggestion more rigorously.
The protein kinase has been purified extensively, but not yet to a homogeneous state. Analysis of the purified samples by SDS-PAGE consistently showed a 43-kDa protein that represented about 50% of the total proteins on the gel. On the basis of a number of observations, it may be concluded that the 43-kDa protein is the Cdk inhibitory kinase. The protein kinase nature of the 43-kDa band was revealed by the ability of the protein to undergo autophosphorylation on SDS-PAGE gel (Fig. 4). The in gel autophosphorylation and protein stain intensity of the 43-kDa band on SDS-PAGE invariably correlated with the protein kinase activity in the Mono Q column eluents, suggesting strongly that this protein kinase is responsible for the Cdk kinase activity (Fig. 3A). Furthermore, we observed that the kinase was inhibited by cAMP and weakly by AMP, and that the in gel autophosphorylation of the 43-kDa protein was inhibited similarly by the nucleotides (Fig. 5).
It should be noticed that the inhibition of the kinase by cAMP requires close to millimolar concentrations of the nucleotide, which are significantly higher than those found in cells. The observation suggests that the inhibition of the kinase by cAMP is not a physiologically relevant effect. On the other hand, the possibility that physiological concentrations of cAMP could affect the kinase under special cellular conditions and/or in concert with other endogenous regulatory factors cannot be excluded. Cdks as well as protein kinases and phosphatases that are involved in regulation of Cdks are all controlled by multiple regulatory factors (for review, see (31) ). How these multiple factors interact to modulate each other's effects is far from clear.
The detection of a Thr-14-specific Cdk inhibitory kinase has made it possible to study the molecular mechanism of the interaction of Thr-14 phosphorylation with other regulatory mechanisms that modulate the activities of Cdks. For example, we have shown that Thr-14 phosphorylation of Cdk5 is independent of the binding of Cdk5 to its specific activator. More importantly, the present study has unambiguously established that phosphorylation of Thr-14 alone is sufficient to bring about close-to-complete inhibition of many cdc2-like kinases (Fig. 2, B and C). The observation strongly supports the ``double block model'' proposed by Krek and Nigg(9) . These investigators observed that overexpression of A14F15 double-site Cdc2 mutant in HeLa cells caused severe premature activation whereas overexpression of single-site Cdc2 mutant of either Ala-14 or Phe-15 had only a mild effect on the cell cycle. Thus, they proposed that phosphorylation of either Thr-14 or Tyr-15 of Cdc2 could restrain the premature activation of Cdc2 kinase.
The phosphorylation on Thr-14 of Cdc2 had been thought to be a unique phosphorylation regulatory mechanism in higher eukaryotes since Cdc2 in yeasts was not found to be phosphorylated on Thr-14. However, Haese et al.(32) recently showed that under certain conditions, such as overexpression of Wee1 kinase, Thr-14 phosphorylation of Cdc2 could be demonstrated in S. pombe. The phosphorylation of Thr-14 in yeasts appears to be dependent on the prior phosphorylation of Tyr-15 and the presence of active Wee1 kinase. Although Mik1 has been suggested to play a redundant role to Wee1 in Tyr-15 phosphorylation in yeasts, overexpression of Mik1 did not result in the phosphorylation of Cdc2 on Thr-14 residue. These results are compatible with the suggestion that Thr-14 phosphorylation in yeasts is catalyzed by Wee1 kinase. In view of the interlocking regulation of cell cycle factors, the possibility that a distinct yeast Thr-14 kinase whose activation is coupled to the activity of Wee1 kinase should also be considered. The identification of the Cdk Thr-14 kinase of the present study in terms of its primary structure may aid in the search for the putative yeast T14 kinase.
The identification and quantification of the Cdk T14 kinase during
purification depended on the use of specific peptide substrates:
cdc2(6-20) and a Tyr-negative peptide analogue (i.e. cdc2(6-20)F15K19). Peptides as modeled kinase substrates
should be applied cautiously. We previously showed that Src family
kinases catalyzed highly efficient phosphorylation of cdc2(6-20)
on Tyr-15 (29, 30) , yet they failed to catalyze the
phosphorylation of Cdc2 protein to any significant extent. ()In the present instance, however, the enzyme purified on
the basis of its peptide phosphorylation activity was found to catalyze
the phosphorylation of relevant protein substrates and to elicit the
expected effect on their activities. A bacterially expressed truncated
form of human Wee1 kinase has been reported to phosphorylate Cdc2
N-terminal peptide(13) , but the intact form of Wee1 kinase has
not been shown to phosphorylate the peptide. The possibility that the
enzyme purified in this study may represent a proteolytically derived
form of the kinase has not been ruled out.
As Nclk is a relatively new addition to the family of Cdc2-like kinases, its regulatory properties have only begun to be investigated (19, 20, 21, 25, 28, 33) . On the basis of sequence homology, it has been suggested that the various regulatory phosphorylation reactions modulating Cdc2 kinase activity may also be involved in the regulation of Nclk activity, for instance, as is the case for Cdk2(34, 35) . However, we have recently shown that the activation of Cdk5 by its specific activator, neuronal Cdk5 activator (Nck5a), is independent of Cdk5 phosphorylation(20, 25) . This is in contrast to a number of well-documented cases of cyclin activation of Cdks where the kinase activation depends on the phosphorylation of the Cdk protein on a specific threonine residue by Cdk activating kinase(22, 23, 24) . On the other hand, the observation that Nclk can be phosphorylated on Thr-14 in vitro by the Cdk T14 kinase to result in the enzyme inactivation supports the suggestion that the mechanisms of negative regulatory phosphorylations are conserved in Nclk.
A number of studies have suggested that phosphorylation of Cdc2 on tyrosine and threonine residues takes place in a cyclin-dependent manner(36, 37, 38, 39) . On the other hand, the phosphorylation of Cdk5 by the purified Cdk T14 kinase could occur in the absence of its partner, Nck5a (Fig. 5). While this apparent difference between Cdc2 and Cdk5 phosphorylation may be attributed to the unique structure of Cdk5 and Nck5a, which shows very limited sequence homology to cyclins, the possibility that it is a result of a difference in experimental conditions should not be overlooked(24) . The control of Cdk activities involves the interplay of various mechanisms including cyclin activation, inhibition by specific protein inhibitors, the activation by Cdk activating kinase-catalyzed Cdk phosphorylation and the inhibitory phosphorylations on Thr-14 and Tyr-15 (reviewed in (31) ). Thus, the question as to the favored conditions of the Cdk phosphorylation by the Cdk T14 kinase has to be addressed by systematic studies of the effect of the various regulatory factors and conditions on the phosphorylation reactions.
The main thrust of the present work is to demonstrate the existence of a novel protein kinase that is capable of in vitro inhibition of a number of Cdc2-like kinases. The general characterizations of the enzyme are compatible with the suggestion that this kinase is the physiological kinase for the Cdk Thr-14 phosphorylation. Future work will have to be carried out toward testing this suggestion and elucidating how this enzyme contributes to the regulation of the Cdc2 family kinases in the cells. It should be noticed that the significance of this kinase may be broader than the regulation of Cdk family kinases. We have carried out gene library search for cdc2(6-20) homologous sequences with serine or threonine at the position corresponding to Thr-14 of Cdc2 and uncovered a large number of protein kinases and other proteins that contain such sequences (results not shown).