(Received for publication, March 5, 1996)
From the
We have purified to near homogeneity from rat brain two
Ca-calmodulin-dependent protein kinase I (CaM kinase
I) activating kinases, termed here CaM kinase I kinase-
and CaM
kinase I kinase-
(CaMKIK
and CaMKIK
, respectively). Both
CaMKIK
and CaMKIK
are also capable of activating CaM kinase
IV. Activation of CaM kinase I and CaM kinase IV occurs via
phosphorylation of an equivalent Thr residue within the
``activation loop'' region of both kinases, Thr-177 and
Thr-196, respectively. The activities of CaMKIK
and CaMKIK
are themselves strongly stimulated by the presence of
Ca
-CaM, and both appear to be capable of
Ca
-CaM-dependent autophosphorylation. Automated
microsequence analysis of the purified enzymes established that
CaMKIK
and -
are the products of distinct genes. In addition
to rat, homologous nucleic acids corresponding to these CaM kinase
kinases are present in humans and the nematode, Caenorhabditis
elegans. CaMKIK
and CaMKIK
are thus representatives of a
family of enzymes, which may function as key intermediaries in
Ca
-CaM-driven signal transduction cascades in a wide
variety of eukaryotic organisms.
Ca-calmodulin (CaM)(
)-dependent
protein kinases (CaM kinases) I and IV are distinguished among members
of the CaM kinase subfamily by their dependence upon phosphorylation by
distinct protein kinases (CaM kinase kinases) for maximal activity (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
Activating phosphorylation occurs at an identically positioned Thr
residue in both cases: Thr-177 in CaM kinase I (8, 13) and Thr-196 in CaM kinase IV(11) ,
although for the latter, Ser phosphorylation in the
NH
-terminal region may also contribute to activation (10, 14) . The activating Thr is three amino acids
NH
-terminal to a highly conserved GTPXXXAPE
sequence present in protein kinase catalytic subdomain
VIII(15) . Phosphorylation in this region, termed the
``phosphorylation lip'' or ``activation loop,'' has
been shown to be essential for the regulation and activity of a number
of protein kinases including protein kinase A(16) , protein
kinase C
II(17, 18) , members of the
mitogen-activated protein kinase
family(19, 20, 21) , and the cyclin-dependent
protein kinases(22, 23, 24) .
In the context of CaM kinase regulation, an important unresolved issue is the number and identity of the CaM kinase kinases. An activating kinase of approximately 52 kDa was purified from pig brain using CaM kinase I as substrate(5, 9) , while one of 66-68 kDa was purified from rat brain using CaM kinase IV as substrate(6, 7) . It was subsequently reported, however, that CaM kinase I kinase purified from pig brain (11) , or an enzyme partially purified from rat brain(13) , was capable of phosphorylating and activating CaM kinase IV. Conversely, a recently cloned and expressed, rat brain CaM kinase IV kinase was found to be capable of activating CaM kinase I(25) . This raised the possibility that a single kinase kinase may be responsible for phosphorylating both CaM kinases I and IV. However, multiple chromatographic peaks of CaM kinase I kinase activity were observed during its purification from pig brain(5) , suggesting that multiple CaM kinase kinases with overlapping substrate specificities (11) could be present.
We have now purified CaM kinase kinases from rat brain using CaM kinase I activation as an assay and report that two such kinases are present, both having the ability to activate CaM kinase I and CaM kinase IV. We also show by direct amino acid sequencing that they are isoenzymes encoded by different genes and are likely to be representatives of a family of enzymes widely distributed among eukaryotic organisms.
The purification of CaM kinase kinases from rat brain
consisted of sequential chromatography on DEAE-Sepharose,
hydroxylapatite, calmodulin-Sepharose, and heparin-Sepharose. The
activity profile of fractions eluted from the heparin-Sepharose column
with a linear salt gradient is shown in Fig. 1. Two clearly
separated activity peaks were observed whether the fractions were
assayed for the ability to activate CaM kinase I or CaM kinase IV. The
virtual superimposition of the profiles obtained using the two kinase
substrates indicates that the same enzymatic species are responsible
for activation of both CaM kinases. When the activating phosphorylation
sites of CaM kinases I and IV, Thr-177 and Thr-196,
respectively(8, 11, 13) , were mutated to
non-phosphorylatable alanine residues, activation by any of the column
fractions was undetectable. This indicates that kinase kinases present
in both peaks activate the two CaM kinases by phosphorylating the
equivalent activation loop Thr residue. Aliquots of each column
fraction were also analyzed by SDS-PAGE and protein staining with
Coomassie Blue R-250 (Fig. 1, lower panel). CaM kinase
kinase activity throughout the first peak correlated with the relative
amounts of a band that electrophoresed slightly faster (M
69,600
) than the BSA standard
whereas activity in the second peak was associated with a more slowly
migrating band (M
73,200
). Note
that some of the most active fractions (e.g. fractions
18-20, 23-25) contain exclusively the
69- or
73-kDa protein, indicating that the the two-peak pattern is not
explainable by carryover of one or the other protein into the adjacent
peak. We have designated the faster migrating band as CaM kinase I
kinase-
(CaMKIK
) and the slower migrating band CaM kinase I
kinase-
(CaMKIK
). Based on the level of purity obtained, the
purification procedure described here is suitable for the preparation
of both CaM kinase kinases to near homogeneity.
Figure 1:
Identification of CaM kinase kinases
separated by heparin-Sepharose column chromatography. CaM kinase
kinases were purified from rat brain as described under
``Experimental Procedures.'' Analysis of the final step of
the purification, heparin-Sepharose column chromatography, is shown. Top panel, CaM kinase activating activities toward CaM kinase
I, CaM kinase IV, CaM kinase I (T177A), and CaM kinase IV (T196A) in
selected fractions as indicated, were measured as described under
``Experimental Procedures'' in the presence of 1 mM Ca-1 µM CaM. wt, wild
type. Bottom panel, aliquots (16 µl) of selected fractions
(indicated by dotted lines) were analyzed by electrophoresis
in a 7.5% polyacrylamide SDS gel and protein staining with Coomassie
Blue R-250 as described under ``Experimental Procedures.''
Protein standards are indicated in the left margin in kDa. Left and right arrows indicate the positions of
CaMKIK
and CaMKIK
, respectively.
In Fig. 1we
have expressed CaM kinase kinase activities as units/µl column
fraction, where 1 unit is defined as 1 pmol of synapsin site 1 peptide
phosphorylated/min/ng of CaM kinase I or IV. The differences in scale
between CaM kinase I and CaM kinase IV activating activities suggest
that both CaMKIK and CaMKIK
prefer CaM kinase I as substrate
relative to CaM kinase IV. It should be noted, however, that in this
coupled assay absolute differences in units can reflect not only the
extent of activation of the CaM kinases but also their maximal peptide
kinase specific activities achievable under these assay conditions and
that the latter may differ between CaM kinases I and IV. Nonetheless
there is precedence for the notion that kinase kinases of this class
while not absolutely substrate-specific are relatively stringent in
their substrate preferences. Hawley et al.(29) demonstrated that the kinase kinase responsible for the
phosphorylation and activation of 5`-AMP-activated protein kinase
(AMPK) is capable of phosphorylating and activating CaM kinase I and
that conversely pig brain CaM kinase I kinase can phosphorylate and
activate AMPK but that in both of these heterologous reactions the rate
of phosphorylation is 2-3 orders of magnitude slower than that of
the corresponding homologous reactions. Whether a similar selectivity
of phosphorylation of CaM kinase I or IV by CaMKIK
and CaMKIK
exists remains to be established through future detailed kinetic
comparisons.
Three lines of evidence indicated that the previously
characterized pig brain CaM kinase I kinase is itself a
Ca-CaM-regulated protein kinase. 1) It demonstrates
Ca
-dependent binding to CaM-Sepharose(5) . 2)
It phosphorylates and activates, in a
Ca
-CaM-stimulated fashion, CaM kinase I
(1-294), a form of CaM kinase I that has lost its CaM-binding
domain through truncation mutagenesis (8) . 3) It
phosphorylates and activates, in a Ca
-CaM-stimulated
fashion, a non-CaM-binding enzyme, AMPK (29) . We therefore
examined whether either or both CaMKIK
and CaMKIK
are
themselves Ca
-CaM-regulated. As shown in Fig. 2by two criteria, the activities of both enzymes are
strongly stimulated by Ca
-CaM. First,
Ca
-CaM enhances the abilities of both CaM kinase
kinases to activate the CaM-independent fragment, CaM kinase I
(1-294) (Fig. 2, top panel), and second, both CaM
kinase kinases appear to autophosphorylate in a
Ca
-CaM-stimulated fashion (Fig. 2, bottom
panel). Consistent with previous reports using pig brain CaM
kinase I kinase(8, 29) , there is detectable, albeit
slight, activity of both CaMKIK
and -
in the absence of
Ca
-CaM. Previous studies have also established that
Ca
-CaM binding to CaM kinase I promotes its
phosphorylation and activation by CaM kinase I kinase by inducing
exposure of Thr-177 (or a conformation favorable for
phosphorylation)(8, 29) . Further studies will thus be
required to assess the relative importance of these dual effects of
Ca
-CaM in promoting phosphorylation and activation of
CaM kinase I by CaMKIK
and -
.
Figure 2:
The activities of CaMKIK and
CaMKIK
are strongly stimulated by Ca
-CaM.
CaMKIK
and -
were purified from rat brain as described under
``Experimental Procedures.'' Analysis of the final step of
the purification, heparin-Sepharose column chromatography, is shown. Top panel, CaM kinase I (1-294) activating activity in
selected fractions was measured as described under ``Experimental
Procedures'' in the presence of 1 mM Ca
-1 µM CaM or absence of
Ca
-CaM and presence of 2 mM EGTA as
indicated. Bottom panel, aliquots (12 µl) of selected
fractions (indicated by brackets) were incubated for 10 min,
at 30 °C, with the following additions as indicated: 0.8 mM CaCl
, 1 µM CaM, or 3 mM EGTA
(Ca
-CaM omitted). All incubations also contained 50
mM Tris, pH 7.6, 0.5 mM DTT, 10 mM MgCl
, and 20 µM [
-
P]ATP (
2
10
cpm/pmol). Reactions were terminated by boiling in a
SDS-
-mercaptoethanol solution and electrophoresed in a 7.5%
polyacrylamide SDS gel. The autoradiogram of the stained and destained
gel is shown. Left and right arrows indicate the
positions of CaMKIK
and CaMKIK
,
respectively.
In order to prove that
CaMKIK and CaMKIK
are distinct proteins, samples of each were
subjected to automated microsequencing as described under
``Experimental Procedures.'' We obtained a sequence of 165
residues of CaMKIK
and 166 residues of CaMKIK
, which, based
on the sequence of the cDNA of CaM kinase IV kinase (CaMKIVK) reported
by Tokumitsu et al.(25) , could represent 33% of the
amino acid sequences of both
and
. The alignment of these
amino acid sequences with that of CaMKIVK is presented in the bottom two lines of Fig. 3. Based on a 99% identity
over 165 residues, CaMKIK
appears to represent a protein product
either identical or highly related to CaMKIVK(25) . On the
other hand, CaMKIK
clearly represents a separate gene product
relative to CaMKIK
or CaMKIVK showing only 76% identity with
CaMKIK
over 67 overlapping residues and 73% identity with CaMKIVK
over 166 overlapping residues. Moreover, these differences are
distributed throughout the homologous regions. Taken together, Fig. 1Fig. 2Fig. 3demonstrate the existence of two
functionally similar but structurally distinct CaM kinase kinases,
CaMKIK
and CaMKIK
.
Figure 3:
Amino acid sequence alignment of CaM
kinase kinases. Peptide sequences of rat brain CaMKIK and
CaMKIK
are aligned with the cDNA-derived amino acid sequence of
CaMKIVK (25) and other homologous nucleic acids detected by
data base search. hBRAIN (assembled overlapping expressed sequence tags
with accession numbers F06422, R50465, H12132, H19394, and H19237) and
R56818 represent partial cDNAs that were sequenced as part of the Human
Genome Project by the Washington University Expressed Sequence Tags
Project. CELC05H8-2 (accession number U11029) is part of a cosmid
containing genomic DNA from the nematode, C. elegans. The
sequences, aligned with the Pileup program (GCG, University of
Wisconsin and (42) ), were formatted with the residues
identical to the CaMKIVK cDNA-derived sequence being shaded.
Amino acids that could not be confidently identified are represented
with an X. A gap inserted into a continuous sequence is
indicated by a dash. Dots represent an absence of
sequence information.
The peptide sequences were used to
search the available protein data bases for previously cloned
homologous nucleic acids using BLAST. This search was conducted before
the paper by Tokumitsu et al.(25) was published, so
only three highly similar sequences were found. These sequences, named
CELC05H8-2, hBRAIN, and R56818, are also presented in Fig. 3. R56818 represents a partial cDNA sequenced as part of
the Human Genome Project by the Washington University Expressed
Sequence Tag Project and cloned by the IMAGE Consortium at the Lawrence
Livermore National Laboratory. hBRAIN is a designation we have given to
a sequence assembled from four overlapping partial human cDNAs (R50465,
H12132, H19237, and H19394), cloned and sequenced as was R56818, and
one additional overlapping clone (F06422). As shown in Fig. 3,
hBRAIN aligns with the NH-terminal portion of CaMKIVK, and
R56818 aligns with the COOH-terminal part without overlap of the two
sequences. We have obtained a human cDNA by screening a brain cDNA
library with sequences derived from those present in R56818. Whereas
the cDNA is not complete, we have obtained enough sequence to ascertain
that hBRAIN and R56818 represent parts of the same mRNA (data not
shown). Based on the data shown in Fig. 3, the human sequence is
much more similar to CaMKIK
(95% identity over 132 overlapping
residues) than to either CaMKIK
(76% identity over 76 overlapping
residues) or CaMKIVK (70% identity over 268 overlapping residues).
Thus, the putative protein encoded by the human nucleic acid appears to
be a homologue of rat CaMKIK
.
The other sequence shown in Fig. 3, CELC05H8-2, is part of a cosmid containing genomic DNA from the nematode, Caenorhabditis elegans. The C05H8 cosmid was cloned and sequenced by the Genome Sequencing Center at Washington University in collaboration with the Sanger Centre in Cambridge, United Kingdom. The putative C. elegans protein kinase is predicted to be comprised of 357 amino acids and would be 56% identical to CaMKIVK over its entire length. The coding sequences were predicted from computer analysis using the Genefinder program. We have used a fragment of the cosmid containing the nematode gene to screen a C. elegans cDNA library. Sequence analysis of the positive clones reveals a region identical to the predicted sequence from Ile-366 to Arg-465. The likely existence of a protein in the nematode that corresponds to a CaM kinase kinase underscores the idea that this newly discovered family of regulatory protein kinases may be widely distributed among eukaryotic organisms.
It may be inferred from our
data that mRNAs for two distinct CaM kinase kinases exist in rat brain.
It seems likely that multiple activating kinases are required to
regulate the activities of CaM kinases I and IV in different cells and
tissues. This idea is consistent with the differential cellular and
tissue distributions of CaM kinases I and IV. Whereas CaM kinase IV
expression is restricted to brain, thymic lymphocytes, and meiotic male
germ
cells(30, 31, 32, 33, 34, 35) ,
CaM kinase I is present in most, if not all, cell
types(8, 12) . Even within the brain, the regions in
which these two CaM kinases are enriched differ markedly. For example,
CaM kinase IV is abundant in the cerebellar granule cells but is
largely absent from most brain stem nuclei(36) . By contrast,
CaM kinase I was not detected in cerebellar granule cells (37) whereas intense immunoreactivity was observed in brain
stem nuclei(38) . At the subcellular level CaM kinase I is
cytosolic (38) whereas CaM kinase IV has a predominantly
nuclear localization(39, 40) . Our results now make it
possible to test, in future studies, whether CaMKIK and -
demonstrate differential tissue and/or subcellular localizations that
correlate with the distribution of either of the CaM kinase targets.
Differences in distribution, combined with the possibility of
tissue-specific mechanisms for initiation of the CaM kinase signal
transduction cascades, suggest that future efforts to understand the
regulation and function of the CaM kinase kinase family of enzymes will
prove to be very rewarding.
Note Added in Proof-While
this paper was under review a paper appeared (Tokumitsu, H., and
Soderling, T. R.(1996) J. Biol. Chem.271, 5617-5622) reporting activation of CaM kinase IV by
[Abstract/Full Text]
phosphorylation of Thr-196 by the rat brain CaM kinase kinase
(CaMKIVK), which appears to correspond to CaMKIK.