From the Department of Biochemistry, Queen's University, Kingston,
Ontario K7L 3N6, Canada and the Department of Physiology
and Biophysics, Case Western Reserve University School of Medicine,
Cleveland, Ohio 44106-4970
Received for publication, October 13, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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Dictyostelium myosin II heavy chain
kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein
kinase catalytic domain that displays no sequence identity to the
catalytic domain present in conventional serine, threonine, and/or
tyrosine protein kinases. Several proteins, including myelin basic
protein, myosin regulatory light chain, caldesmon, and casein were
phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C
catalytic domains. Phosphoamino acid analyses of the proteins showed
that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further
examined using a synthetic peptide library
(MAXXXX(S/T)XXXXAKKK; where X is
any amino acid except cysteine, tryptophan, serine, and threonine and
position 7 contains serine and threonine in a 1.7:1 ratio).
Phosphorylation of the peptide library with the three MHCK catalytic
domains resulted in 97 to 99% of the phosphate being incorporated into
threonine, while phosphorylation with a conventional serine/threonine
protein kinase, the p21-activated kinase, resulted in 80% of the
phosphate being incorporated into serine. The acceptor amino acid
specificity of MHCK A was tested directly by substituting serine for
threonine in a synthetic peptide and a glutathione
S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower
rate than the threonine-containing substrates. The results indicate
that the MHCKs are specific for the phosphorylation of threonine.
Dictyostelium discoideum myosin II heavy chain kinase A
(MHCK A)1 phosphorylates
threonine residues at positions 1823, 1833, and 2029 in the Truncation analysis of MHCK A shows that the central 35-kDa domain, in
the absence of both the coiled-coil domain and WD-repeat domain, is a
highly active protein kinase, with the ability to catalyze phosphoryl
transfer from ATP to both peptide and protein substrates (9). Of
considerable interest is the finding that the MHCK A catalytic domain
shares no significant sequence similarity to the conserved catalytic
domain that distinguishes the extremely large family of
"conventional" serine, threonine, and/or tyrosine protein kinases
or to the catalytic domain present in the prokaryotic and eukaryotic
histidine kinases (9-11).
A domain homologous to the MHCK A catalytic domain has now been
recognized in several additional proteins, including two
Dictyostelium proteins: MHCK B (12) and MHCK C, the product
of the mhkC gene (GenBankTM accession
number 3420749). MHCK B and MHCK C are structurally similar to
MHCK A, consisting of a catalytic domain followed by a WD-repeat
domain. However, neither MHCK B nor MHCK C has the extended N-terminal
coiled-coil domain found in MHCK A. In vitro studies show
that bacterially expressed MHCK B is an active protein kinase and that
it phosphorylates the same sites in the Dictyostelium myosin
II tail as MHCK A (12). Although the in vivo role of MHCK B
remains to be determined, these studies suggest that MHCK B may
cooperate with MHCK A to regulate myosin II filament formation. No
information is available concerning the enzymatic activity or potential
substrates of MHCK C.
A number of mammalian proteins with a domain homologous to the MHCK A
catalytic domain have also been identified (13, 14). The best
characterized of these proteins is the eukaryotic elongation factor-2
(eEF-2) kinase, which inhibits the elongation phase of protein
synthesis by phosphorylating two threonine residues in eEF-2 (15).
Site-directed mutagenesis and truncation analysis of the eEF-2 kinase
shows that a pair of conserved cysteine residues within the MHCK
A-related kinase domain as well as sequences C-terminal to this domain
are required for activity (16).
It is now clear that MHCK A represents the founding member of a novel
family of protein kinases. Studies on the MHCK A family of protein
kinases have so far failed to reveal any unusual or distinct catalytic
properties that would serve to distinguish them from the superfamily of
conventional serine/threonine protein kinases. In this paper we show
that the MHCK A, MHCK B, and MHCK C catalytic domains are remarkably
specific for the phosphorylation of threonine residues in both peptide
and protein substrates.
Reagents--
ATP, myelin basic protein (MBP), and bovine casein
were from Sigma and [ Plasmid Constructs--
All DNA manipulations were carried out
using standard methods. The MHCK A (GenBankTM accession
number 1170675) catalytic domain used in these studies, designated
A-CAT, comprises residues 552-841 of MHCK A and has a C-terminal
hexahistidine tag. A-CAT is identical to the previously described T-5
(9). A vector expressing the MHCK B (GenBankTM accession
number 3122317) catalytic domain was made by excising a
1.4-kilobase insert fragment from pMM3.1, which contains the full-length cDNA coding region of MHCK B, using an internal
EcoRV site (nucleotides 1391) and an XbaI site in
the upstream polylinker of pMM3.1. The XbaI site was blunted
by treatment with Klenow polymerase and then ligated into pGEX-2T
vector that had been digested with EcoRI and made blunt.
Digestion of this intermediate with EcoRI and subsequent
recircularization yielded a vector that expressed residues 13-459 of
MHCK B, designated B-CAT, fused in-frame to the C terminus of GST. The
genomic clone of MHCK C (GenBankTM accession number
3420749) contains introns, so a cDNA clone corresponding to MHCK C
was isolated from a 4-h developed Dictyostelium Protein Expression and Purification--
A-CAT, B-CAT, and C-CAT
were expressed in Escherichia coli BL21(DE3). Bacteria were
grown overnight at 37 °C to an A600 of 0.6-1.0 and cooled to 25 °C prior to induction with
isopropyl- Peptide Synthesis--
MH-3S, a variant of the previously
described 16-residue peptide MH-3 (20), was synthesized using the
HBTU/HObt coupling protocols on an Applied Biosystems 431A peptide
synthesizer in the Department of Biochemistry, Queen's University. The
same coupling protocols were used to construct a degenerate peptide
library with the sequence MAXXXX(S/T)XXXXAKKK,
where S/T designates a position containing a mixture of serine and
threonine and X indicates a position containing all the
common amino acids except tryptophan, cysteine, serine, and threonine
(21). Position seven was synthesized using equal moles of
N Kinase Reactions--
In vitro kinase assays for
A-CAT, B-CAT, and C-CAT were carried out at 25 °C in kinase buffer
(2 mM MgCl2, 1 mM dithiothreitol, 0.25 mM [ Phosphopeptide Separation and Sequence Analysis--
The peptide
library (3.3 mg/ml) was incubated in 300 µl of kinase buffer with
A-CAT, B-CAT, or C-CAT. The reaction was stopped by addition of 300 µl of acetic acid and the phosphorylated peptides isolated as
described (21, 22). Briefly, the mixture was passed through a 10,000 molecular weight cut-off Ultrafree-4 Centrifugal Filtration Unit to
remove the kinase and chromatographed on a DE-53 column equilibrated in
30% acetic acid to remove [ Phosphoamino Acid Analysis--
Radiolabeled proteins were
separated by SDS-polyacrylamide gel electrophoresis and transferred to
Immobilon P membrane (Millipore). After staining with Amido Black, the
piece of membrane containing the protein band was excised, washed with
water and methanol, and hydrolyzed in 6 N HCl at 110 °C
for 2 h under vacuum (24). Peptides were separated from kinase
using a 10,000 molecular weight cut-off Ultrafree-4 Centrifugal
Filtration Unit, chromatographed over DE-53 cellulose equilibrated in
30% acetic acid to remove [ Expression of the Catalytic Domains of MHCK A, MHCK B, and MHCK
C--
The atypical protein kinase catalytic domain first defined in
Dictyostelium MHCK A is present in two related
Dictyostelium proteins: MHCK B and MHCK C (Fig.
1A). For the studies reported here the isolated catalytic domains of MHCK A, MHCK B, and MHCK C,
designated here as A-CAT, B-CAT, and C-CAT, respectively, were expressed in bacteria. A-CAT comprises residues 552-841 of MHCK A,
B-CAT comprises residues 13-459 of MHCK B, and C-CAT comprises residues 19-283 of MHCK C. A-CAT, B-CAT, and C-CAT were insoluble if
expressed at 37 °C, but soluble if expressed at 25 °C. A-CAT and
C-CAT, which were expressed with a hexahistidine tag, could be purified
with yields of 1-2 mg/liter of culture while B-CAT, which was
expressed as a GST fusion protein, was obtained with a yield of ~0.1
mg/liter of culture (Fig. 1B).
Phosphorylation of Peptide and Protein Substrates--
The
16-residue peptide MH-3 (RKKFGEAEKTKAKEFL-amide) has been
previously described (20) and is based on the MHCK A target site in the
Dictyostelium myosin II tail at residue 2029 (underlined in
peptide above). MH-3 (and its variants) are presently the only documented peptide substrates for MHCK A and MHCK B (12, 20, 27).
Measurement of the initial rates of phosphorylation showed that MH-3 is
a much better substrate for A-CAT and B-CAT than for C-CAT (Table
I). MBP has previously been identified as
a good substrate for A-CAT (9) and is also phosphorylated at a high
rate by B-CAT and C-CAT. Several other protein substrates, including
casein, RLC, and caldesmon were found to be substrates for A-CAT,
B-CAT, and C-CAT, although they were phosphorylated at a considerably
lower rate than MBP or MH-3 (Table I). When the kinase reactions were
allowed to proceed to completion, the maximal amount of 32P
incorporated by A-CAT, B-CAT, and C-CAT into individual proteins was
often quite different, suggesting that the sites targeted by each
catalytic domain are not identical (Table I).
Proteins that had been maximally phosphorylated by A-CAT were subjected
to acid hydrolysis and their phosphoamino acid content examined by
two-dimensional electrophoresis on thin-layer cellulose plates. In all
cases the phosphorylated proteins contained significantly greater
amounts of Thr(P) than either Ser(P) or Tyr(P) (Fig.
2). Measurement of the amount of
radioactivity at the position corresponding to each of the phosphoamino
acid standards indicated that Tyr(P) accounted for less than 1%,
Ser(P) for 2 to 5%, and Thr(P) for 95 to 98% of the total
radioactivity (Table I). Since the level of phosphate incorporated into
tyrosine residues was negligible, further analysis focused on
quantifying the relative amounts of Ser(P) and Thr(P). Experiments in
which Ser(P) and Thr(P) were separated by one-dimensional
electrophoresis at pH 1.9 showed that B-CAT and C-CAT displayed a
strong preference for the phosphorylation of threonine residues in
caldesmon (Fig. 3A) and other
protein substrates (Table I).
The incorporation of phosphate into A-CAT, B-CAT, and C-CAT as a result
of autophosphorylation was also examined (9, 12). A-CAT incorporated 2 mol of phosphate/mol, B-CAT 10 mol of phosphate/mol, and C-CAT only 0.5 mol of phosphate/mol. Phosphoamino acid analysis showed that Thr(P)
accounted for 98%, 80 and 90% of the total phosphate incorporated
into A-CAT, B-CAT, and C-CAT, respectively (Fig. 3B).
Phosphorylation of a Serine/Threonine Peptide Library--
To
further investigate the acceptor amino acid specificity of A-CAT,
B-CAT, and C-CAT a degenerate serine/threonine peptide library was
synthesized composed of peptides with the sequence MAXXXX(S/T)XXXXAKKK, where X
represents a degenerate position containing all the amino acid except
cysteine, tryptophan, serine, and threonine and position seven contains
a mixture of serine and threonine. This peptide library provides a
choice of more than 4 × 109 distinct peptides each
with a single serine or threonine residue as a potential site of
phosphorylation. Although the serine/threonine position was synthesized
using equal moles of N
The serine/threonine peptide library was phosphorylated using A-CAT,
B-CAT, C-CAT, or the conventional serine/threonine protein kinase PAK.
The reactions were terminated when 1% of the peptides were
phosphorylated, so that the kinases would not be forced to phosphorylate suboptimal substrates (21). Phosphoamino acid analysis of
the peptide library showed that A-CAT, B-CAT, and C-CAT incorporated
phosphate almost exclusively into threonine residues, while PAK
predominately phosphorylated serine residues (Fig.
4, A and B).
Quantification of the results demonstrated, remarkably, that 97-99%
of the total phosphate incorporated by A-CAT, B-CAT, and C-CAT was
recovered as Thr(P) (Table I). In contrast, 80% of the phosphate
incorporated by PAK was recovered as Ser(P).
The phosphoamino acid analyses results reported above were obtained
following 2 h of acid hydrolysis. The half-life of Thr(P) is
greater than that of Ser(P) in 6 N HCl at 105 °C (more
than 25 h as compared with 8 h) suggesting that shorter
hydrolysis times might increase the ratio of Ser(P) to Thr(P) (29). To examine this possibility, the peptide library phosphorylated by A-CAT
was subjected to acid hydrolysis times varying from 30 min to 4 h.
At 30 min the amount of Ser(P) was still less than 4% that of Thr(P)
(Fig. 5).
Consensus Phosphorylation Sequences for A-CAT, B-CAT, and
C-CAT--
The peptide library was phosphorylated to a low level
(~1% of the peptides phosphorylated) using A-CAT, B-CAT, or C-CAT.
The small fraction of phosphopeptides was isolated from the bulk of unphosphorylated peptides as described under "Materials and
Methods" and subjected to amino acid sequence analysis to determine
the amino acid composition at each degenerate position. The abundance of individual amino acids at the eight degenerate positions in the pool
of phosphorylated peptides was then compared with the abundance of the
same amino acid at the same position in the starting peptide library to
give a selectivity value (Table II). A
selectivity value above 1 indicates that peptides with that residue
have been preferentially chosen by the kinase as substrates during the
phosphorylation reaction (22). The results showed that A-CAT, B-CAT,
and C-CAT all favor peptides with tyrosine at the
The consensus recognition sequence determined for A-CAT (YAYDTRYRR) was
synthesized and found to be a good A-CAT substrate. Kinetic analysis
showed that A-CAT displayed a Km for YAYDTRYRR of
550 µM and a kcat of 14 s Substitution of Serine for Threonine in Substrates--
To
directly examine the impact that a threonine or serine residue at the
site of phosphorylation has on the activity of A-CAT, the
serine-containing counterpart of MH-3 was synthesized (MH-3S: RKKFGEAEKSKAKEFL-amide). In contrast to MH-3, MH-3S was not appreciably phosphorylated by A-CAT even at a concentration of 2 mM
(Fig. 6B). A second pair of serine/threonine substrates was
prepared by fusing either YAYDTRYRR or YAYDSRYRR to the C terminus of
GST. GST alone was not a substrate for A-CAT (data not shown) but the GST-YAYDTRYRR fusion peptide was readily phosphorylated by A-CAT to a
level of 1 mol of phosphate/mol (Fig. 6C). In contrast, the GST-YAYDSRYRR fusion peptide was a very poor substrate for A-CAT. Over
the linear portion of the time course the threonine-containing GST
fusion peptide was phosphorylated at a rate more than 25-fold higher
than its serine-containing analogue (Fig. 6C). These results directly demonstrate that A-CAT strongly prefers a threonine residue at
the site of phosphorylation.
Dictyostelium MHCK A, MHCK B, and MHCK C contain a type
of protein kinase catalytic domain whose primary sequence is very different from the catalytic domain found in conventional serine, threonine, and/or tyrosine protein kinases (9, 12). The goal of the
present study was to examine the intrinsic substrate specificity of the
MHCK catalytic domains. For this purpose the isolated catalytic domains
of MHCK A, MHCK B, and MHCK C, designated A-CAT, B-CAT, and C-CAT, were
expressed in bacteria and purified (Fig. 1). The use of all three
catalytic domains, which share between 48 and 54% sequence similarity,
provides a degree of confidence that the results obtained should be
applicable to the entire family of MHCK A-related kinases.
A-CAT, B-CAT, and C-CAT phosphorylated a selection of structurally
diverse proteins, including MBP, RLC, caldesmon, and casein, and in
many instances incorporated more than 1 mol of phosphate/mol into these
proteins (Table I). Phosphoamino acid analysis produced the striking
observation that for all of the protein substrates more than 95% of
the phosphate was incorporated into threonine residues with the
remainder being incorporated into serine. No phosphorylation of
tyrosine was observed. Experiments using a degenerate serine/threonine
peptide library showed that when confronted with a choice of a serine
or threonine phosphorylation sites, A-CAT, B-CAT, and C-CAT
overwhelmingly chose peptides containing threonine. As judged by the
distribution of radioactivity following phosphoamino acid analysis,
98-99% of the phosphate incorporated into the peptide library by
A-CAT, B-CAT, and C-CAT was present on threonine residues.
The ability of A-CAT to select between a serine and threonine acceptor
amino acid was further examined using two defined substrates: a
synthetic peptide (MH-3) and a GST fusion peptide. Substitution of
serine for the target threonine in MH-3 and the GST fusion peptide
virtually eliminated phosphorylation by A-CAT (Fig. 6, A and
B). MH-3S, the serine version of MH-3, was such a poor
substrate for A-CAT that its kinetic constants could not be calculated; thus, it was not possible to determine whether the low rate of phosphorylation of MH-3S was due to its inability to bind to A-CAT or
to a large decrease in kcat. However, when added
to phosphorylation reactions containing MH-3, MH-3S did not behave as a
competitive inhibitor (data not shown). These results suggest that the
replacement of threonine with serine substantially reduces the affinity
of the peptide for A-CAT.
A-CAT, B-CAT, and C-CAT are not absolutely specific for threonine since
detectable levels of Ser(P) were obtained following the phosphoamino
acid analysis of protein and peptide substrates. The highest proportion
of Ser(P), amounting to 20% of the total phosphoamino acids, was
present in the autophosphorylated B-CAT (Table I). A possible
explanation for this relatively high level of serine phosphorylation is
that B-CAT autophosphorylates multiple sites via an intramolecular
reaction. If this is the case, relatively unfavorable serine residues
may be phosphorylated simply because they are presented to the active
site of B-CAT at a very high effective concentration. Interestingly, it
has been reported that the mutation of the target threonine residues in
the Dictyostelium myosin II tail to serine residues does not
prevent phosphorylation by partially purified MHCK A (2). This result
seems unusual given the threonine specificity for the MHCK A catalytic
domain documented here, but may possibly be explained by the finding that MHCK A is physically targeted to myosin II by the WD-repeat domain
(8, 27). By tethering the MHCK A catalytic domain in close proximity to
the serine residues, the WD-repeat domain may enhance their phosphorylation.
The strong preference to phosphorylate threonine residues in both
peptides and proteins distinguishes the MHCK A family of protein
kinases from conventional protein serine/threonine kinases. Indeed,
many conventional serine/threonine kinases display a bias toward the
phosphorylation of serine residues. A compilation of the sites
phosphorylated in proteins by conventional kinases shows that serine
residues are targeted about four times more frequently than threonine
residues (30). A comparable distribution (80% serine and 20%
threonine) was obtained in this study when PAK, a conventional protein
kinase, was used to phosphorylate the serine/threonine peptide library
(Table I). Peptides containing serine residues are more effective
substrates than their threonine-containing counterparts for several
conventional protein kinases, including the cAMP-dependent
protein kinase (31-34). Assays with the
cAMP-dependent protein kinase, for example, show that
the replacement of serine in Kemptide (LRRASLG) with threonine produces
a 5-fold decrease in kcat and a 20-fold increase
in Km (31, 35).
At least for some substrates, the active site of conventional protein
kinases does not seem to readily accommodate a methyl group on the
The catalytic domain of the human eEF-2 kinase exhibits 40-46%
sequence similarity to the Dictyostelium MHCKs, suggesting that it is likely to display the same fundamental properties, including
a specificity for threonine residues, as the MHCKs. Consistent with
this proposal, the sites phosphorylated by the eEF-2 kinase in
elongation factor-2 are Thr-56 and Thr-58 (15). The eEF-2 kinase
autophosphorylates on serine residues (47, 48) but, as noted above,
intramolecular autophosphorylation sites may not be representative of
the sites phosphorylated on optimal exogenous substrates. Clearly, it
will be important to directly test the acceptor amino acid specificity
of the eEF-2 kinase and other mammalian members of the MHCK A family.
Conventional protein kinases recognize phosphorylation sites located
within the context of a characteristic sequence of amino acids and it
seems likely that the same is true for A-CAT, B-CAT, and C-CAT. Based
on the observation that MHCK A phosphorylates sites in the The three MHCK catalytic domains phosphorylate individual substrates at
significantly different rates and also incorporate significantly
different amounts of phosphate, indicating that they have distinct
substrate specificities (Table I). An initial attempt to define the
consensus sequences recognized by A-CAT, B-CAT, and C-CAT was made
using the degenerate peptide library method, which tends to select for
peptide substrates that have low Km values or high
kcat/Km ratios (21, 22). The
results indicate that all three catalytic domains share some common
recognition elements, including a preference for tyrosine in the It is of interest to compare the consensus phosphorylation sequence
predicted using the peptide library with the sequences of sites
phosphorylated in protein substrates; however, no MHCK C protein target
sites have been mapped, and the only identified sites for MHCK A and
MHCK B are the three threonine residues in the Dictyostelium
myosin II tail (1, 2, 12). A part of the predicted A-CAT consensus
sequence (DT-basic-Y-basic) corresponds exactly to the
sequence of the 1833 site (DTKYK) within the myosin II tail
but is less similar to the 2029 site (KTKTK) and the 1823 site (ATKTQ) (phosphorylated residue is underlined). (1).
The predicted consensus sequence for B-CAT (RTV-basic-basic) is most similar to the sequence of the 2029 site. The relative rates at which
A-CAT and B-CAT phosphorylate the three sites in the myosin II tail is,
however, not known.
In summary, the studies reported here provide strong evidence that the
MHCK A family of protein kinases exhibit a specificity for threonine
residues much greater than that usually associated with conventional
serine/threonine protein kinases. Since protein kinases are classified
based on the nature of the acceptor amino acid (54), we propose that
the MHCK A-related kinases be classed as threonine kinases. The ability
to target threonine residues provides a rationale for why the MHCK A
catalytic domain has been conserved throughout evolution (albeit in a
relatively small group of proteins) and is likely to be critical for
understanding how these kinases function in signaling pathways. The
unique acceptor amino acid specificity of the MHCK A-related kinases
might also make them useful reagents in cases where the selective
phosphorylation of threonine residues in peptides or proteins is desired.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical
coiled-coil tail of myosin II (1, 2). Phosphorylation of the threonine
residues drives myosin II from a filamentous to a monomeric state and
plays a central role in inhibiting the cellular activity of myosin II
(3-6). Sequence analysis of the 130-kDa MHCK A shows that it is
composed of an ~50-kDa N-terminal
-helical coiled-coil domain, a
35-kDa central domain, and a C-terminal WD-repeat domain (7). Native
MHCK A is a multimer with an apparent molecular mass in excess of 1000 kDa by gel filtration analysis and, when visualized by electron microscopy, appears as a highly asymmetric molecule with several globular domains clustered at one end of a 50-nm long rod (8).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was from PerkinElmer
Life Sciences. Caldesmon and myosin regulatory light chain (RLC) were
purified from chicken gizzard as described (17, 18). A plasmid
expressing the full-length, constitutively active mouse p21-activated
kinase (PAK) as a GST fusion protein was a gift of Dr. S. Bagrodia and
has been previously described (19).
GT11 library using a polymerase chain reaction-generated probe. The catalytic domain was amplified from the cDNA clone by polymerase chain reaction using primers that contained BamHI
restriction sites upstream and downstream of the predicted catalytic
domain. The polymerase chain reaction fragment was cloned into the
vector pRSET-A (Invitrogen) and a stop codon was placed downstream by subsequent addition of a 3'-oligonucleotide. The resultant plasmid, pRSET-DG2, expresses a fusion protein containing residues 19-283 of
MHCK C, designated C-CAT, with 36 vector-derived residues at the N
terminus (including the hexahistidine tag) and 5 vector-derived amino
acids at the C terminus of the protein. Two GST fusion peptide expression vectors were prepared by cloning annealed complementary oligonucleotides encoding the desired peptides into the
BamHI/XhoI site of pGEX-4T-3 (Amersham Pharmacia
Biotech). The expressed proteins consist of GST followed by either
YAYDTRYRR or YAYDSRYRR and then 7 vector-derived residues
(LERPHRD). All constructs were confirmed by restriction digest and
DNA sequencing.
-D-thiogalactosidase (1 mM final
concentration). A-CAT and C-CAT were isolated from the 15,000 × g supernatant fraction of sonicated cell lysates using
nickel chelation chromatography according to standard protocols provided with the His-Bind resin (Novagen). C-CAT recovered from the
His-Bind resin was not further purified and was immediately stored in
aliquots at
80 °C. A-CAT was dialyzed against 50 mM NaCl, 20 mM Tris, pH 7.5, and applied to a DE-53 (Whatman)
column equilibrated in the same buffer. The flow-through was collected and passed over an SP Sepharose Fast Flow (Amersham Pharmacia Biotech)
column equilibrated in 50 mM NaCl, 20 mM Tris,
pH 7.0. The flow-through was collected, concentrated using a 10,000 molecular weight cut-off Ultrafree-4 Centrifugal Filtration Unit
(Millipore), and stored at
80 °C. B-CAT, expressed as a GST fusion
protein, was isolated from the 15,000 × g supernatant
of sonicated cell lysates using glutathione-Sepharose beads (Amersham
Pharmacia Biotech). Following elution with buffer containing 30 mM glutathione B-CAT was dialyzed against 12.5 mM Tris, pH 7.5, in 40% glycerol and stored at
80 °C.
GST-peptide fusion proteins were expressed at 37 °C in E. coli DH5
and recovered using glutathione-Sepharose beads.
-FMOC-blocked serine and threonine at 4-fold
excess to the coupling resin while positions denoted by an X
were synthesized using equal moles of a mixture of 16 N
-FMOC-blocked amino acids at 4-fold excess
to the coupling resin. Tryptophan and cysteine were omitted from the
degenerate positions to avoid problems with sequencing and oxidation
while threonine and serine were omitted to ensure that each peptide has
only a single potential site of phosphorylation (21, 22). Sequence analysis of the library, performed at the Alberta Peptide Institute (Edmonton, Alberta), showed that the amount of each of the 16 amino
acids at each degenerate position was consistent from position to
position and varied by no more than a factor of 2 from the expected
value of 6.6% of the total amino acids. Isoleucine and lysine were
present in the lowest amounts (3.0-3.5% of total amino acids) while
proline and glycine were present in the highest amounts (9.5-10.5% of
total amino acids).
-32P]ATP (2-5 × 102 cpm/pmol) and 20 mM TES, pH 7.0). Assays
were initiated by addition of the substrate (concentrations are
provided in Table I and figure legends) followed by 1-5 µg/ml
kinase. In some cases the kinases were preincubated in kinase buffer
for 30 min at 25 °C prior to use. Phosphorylation rates were
determined by removing 40-µl aliquots of the reaction mixture at time
points from 30 s to 6 min. Incorporation of 32P into
MH-3, MH-3S, or MBP was determined by spotting aliquots onto 2 × 2-cm squares of P81 phosphocellulose paper (Whatman) (23). After
washing in 1% phosphoric acid, the paper squares were placed into
liquid scintillation fluid and counted using a Beckman LS 7500 scintillation counter. Assays with caldesmon, casein, and RLC were
stopped by the addition of a one-fifth volume of SDS gel sample buffer
(final concentration 1% SDS, 60 mM Tris-HCl, pH 6.8, 0.2%
-mercaptoethanol, and a trace of bromphenol blue) followed by
boiling for 5 min. Assays with GST peptide fusion proteins were
terminated by addition of 10 µl of glutathione-Sepharose. The resin
was pelleted by centrifugation, washed with phosphate-buffered saline,
and boiled for 5 min in SDS gel sample buffer. Radiolabeled proteins
were separated on SDS-polyacrylamide gel electrophoresis and visualized
and quantified using a storage phosphor screen and a Bio-Rad Personal
Molecular Imager FX. In some cases the protein band of interest was
excised from the gel, placed in liquid scintillation fluid, and counted
in a scintillation counter.
-32P]ATP. Fractions
containing peptides were lyophilized, dissolved in 1 M
NaCl, 50 mM MES, pH 5.5, and applied to a 1-ml column of ferric iminodiacetic acid beads (Pierce) equilibrated in the same buffer. After washes with equilibration buffer and 2 mM
MES, pH 6.0, the phosphopeptides were eluted with 0.5 M
NH4HCO3, pH 8.0. A control experiment was
performed in which the peptide library was incubated in the absence of
kinase and subjected to the same isolation protocol. The peptide
mixtures recovered from the ferric column were sequenced at the Alberta
Peptide Institute. The abundance of each amino acid at each degenerate
position in the phosphopeptide fractions was first corrected for the
presence of non-phosphorylated peptides, the level of which was
estimated by the amount of serine and threonine recovered at cycle 7. The non-phosphorylated peptide control mixture was enriched in aspartic
acid and glutamic acid, and so the major effect of this correction was
to reduce the levels of these two residues (21). The corrected
phosphopeptide data was then divided by the relative abundance of each
amino acid at each degenerate position in the starting mixture.
The abundance ratios were added and normalized to 16 (the number of
amino acids at each degenerate position) to obtain the selectivity
values. Selectivity values above 1 are obtained when an amino acid at a
given position is enriched in the phosphopeptide fraction relative to
the starting mixture and indicates that peptides containing this
residue are preferentially phosphorylated by the kinase (21).
-32P]ATP, dried in a
Speed-Vac vacuum concentrator, and hydrolyzed as described above.
Hydrolysates were resuspended in 1 ml of water and dried in a Speed-Vac
vacuum concentrator. This process was repeated twice, then each
hydrolysate was resuspended in 5-10 µl of pH 1.9 buffer (2.5%
formic acid and 7.8% acetic acid) containing 0.1 mg/ml each of
unlabeled Ser(P), Thr(P), and Tyr(P) standards (Sigma) and
applied to a thin-layer cellulose chromatography plate (20 × 20 cm, Eastman). Electrophoresis was performed using the pH 1.9 buffer
and, if necessary, in a second dimension with pH 3.5 buffer (5% acetic
acid and 0.5% pyridine), using an HTLE-7000 thin layer electrophoresis
unit (C.B.S. Scientific Co.) (25, 26). Phosphoamino standards were
located by reaction with 0.25% (w/v) ninhydrin in acetone, while
32P-labeled Ser(P) and Thr(P) were detected and quantified
using a storage phosphor screen and a Bio-Rad Personal Molecular Imager FX.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of the MHCK catalytic
domains. A, a schematic diagram showing the domain
structure of MHCK A (GenBankTM accession number 1170675),
MHCK B (GenBankTM accession number 3122317), and MHCK C
(GenBankTM accession number 3420749). MHCK A consists of an
-helical coiled-coil domain (dark gray), a novel type of
protein kinase catalytic domain (black), and a WD-repeat
domain (light gray). MHCK B and MHCK C possess a catalytic
domain and WD-repeat domain structurally related to those of MHCK A but
lack an extended coiled-coil domain. B, a Coomassie
Blue-stained SDS-polyacrylamide gel of the bacterially expressed MHCK
catalytic domains used in this study. A-CAT and C-CAT were expressed
with a hexahistidine tag while B-CAT was expressed as a GST fusion
protein.
Substrate phosphorylation and phosphoamino acid analysis
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Fig. 2.
Phosphoamino acid analysis of proteins
phosphorylated by A-CAT. The panels show autoradiographs of the
phosphoamino acid content of: A, RLC; B, MBP;
C, casein; and D, caldesmon, after being
maximally phosphorylated by A-CAT. Following phosphorylation the
proteins were subjected to SDS-gel electrophoresis, transferred to
Immobilon-P, and hydrolyzed to their constituent amino acids. The
hydrolysates were separated by electrophoresis at pH 1.9 and then at pH
3.5 in the directions indicated by the arrows for
panel C. The positions of the unlabeled Ser(P), Thr(P), and
Tyr(P) standards were visualized by ninhydrin staining and are shown as
dotted outlines.
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Fig. 3.
Phosphoamino acid analysis of caldesmon and
autophosphorylated A-CAT, B-CAT, and C-CAT. The panels show
autoradiographs of the phosphoamino acid content of: A,
caldesmon maximally phosphorylated with A-CAT, B-CAT, and C-CAT; and
B, autophosphorylated A-CAT, B-CAT, and C-CAT. Following
phosphorylation the proteins were subjected to SDS-gel electrophoresis,
transferred to Immobilon-P, and hydrolyzed to their constituent amino
acids. Ser(P) and Thr(P) were separated by electrophoresis at pH 1.9. The dotted circles represent the positions of the unlabeled
Ser(P) and Thr(P) standards detected by ninhydrin staining.
-Fmoc-blocked serine
and threonine, quantitative amino acid analysis of the library yielded
values of 0.62 mol/mol for serine and 0.36 mol/mol for threonine
(corrected for 90% recovery after acid hydrolysis). The low abundance
of threonine in the peptide library may reflect the fact that the
-substituted threonine couples at a slower rate than serine during
solid phase peptide synthesis (28).
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Fig. 4.
Phosphoamino acid analysis of the
serine/threonine peptide library. A, an autoradiograph
showing the phosphoamino acid content of the serine/threonine peptide
library following phosphorylation with A-CAT. Peptides were hydrolyzed
to yield free amino acids as described under "Materials and
Methods" and the hydrolysate was subjected to electrophoresis at pH
1.9 and then at pH 3.5 on a thin-layer cellulose plate as indicated by
the arrows. The dotted circles show the positions
of the unlabeled Ser(P), Thr(P), and Tyr(P) standards detected by
ninhydrin staining. B, an autoradiograph showing the
phosphoamino acid content of the serine/threonine peptide library
following phosphorylation with PAK, A-CAT, B-CAT, or C-CAT. Separation
was performed in one dimension at pH 1.9. The dotted circles
represent the positions of the unlabeled Ser(P) and Thr(P) standards
detected by ninhydrin staining.
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Fig. 5.
Acid hydrolysis time course. The
serine/threonine peptide library was phosphorylated with A-CAT.
Phosphoamino acid analysis was performed as described under
"Materials and Methods" except that the duration of the acid
hydrolysis was varied from 30 min to 6 h. Following
electrophoresis on thin-layer cellulose plates at pH 1.9 the amount of
radioactivity in the Pi ( ), Thr(P) (
), and Ser(P)
(
) spots and at the origin (
) was quantified using a Bio-Rad
Personal Molecular Imager FX.
2 and
4 positions
and basic residues at the +3 and +4 positions. In the +1 position A-CAT
displayed a preference for a basic residue and B-CAT strongly selected
for valine. C-CAT showed an unusual preference for aromatic, hydrophobic residues in all positions from
4 to +2.
Substrate specificities of the MHCK catalytic domains
1 (Fig. 6A).
With MH-3 as the substrate, A-CAT exhibited a somewhat lower
Km (280 µM) but an 8-fold lower
kcat (1.8 s
1) (Fig.
6B). Similar kinetic constants for the phosphorylation of
MH-3 by A-CAT have been reported previously (9).
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Fig. 6.
Phosphorylation of peptides and GST
fusion peptides by A-CAT. A, phosphorylation of the
peptide YAYDTRYRR by A-CAT. Rates of phosphorylation at each peptide
concentration were determined by removing aliquots from the reaction at
1, 2, 3, 4, and 5 min and spotting them onto phosphocellulose paper as
described under "Materials and Methods." Over this time period
linear rates of phosphate incorporation were obtained at all peptide
concentrations. The data were fit to a hyperbolic equation by
regression analysis using the program SigmaPlot for Windows (SPSS
Science Inc.). B, phosphorylation by A-CAT of the
peptides MH-3 ( ) and MH-3S (
). The sequence of MH-3S
(RKKFGEAEKSKAKEFL-amide) is identical to that of MH-3 but with serine
in place of threonine. Rates of phosphorylation and data analysis was
performed as described above. C, phosphorylation
by A-CAT of YAYDTRYRRL (
) or YAYDSRYRRL (
) fused to the C
terminus of GST. The inset shows the Coomassie Blue-stained
gel (CB) and the corresponding autoradiograph
(AR) for the threonine (T) and serine
(S) GST peptide fusions phosphorylated for 10, 20, and 40 min. Assays contained 1 mg/ml GST-peptide fusion protein and 5 µg/ml A-CAT. Phosphate incorporation into the substrates was
quantified using a Bio-Rad Personal Molecular Imager FX.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-carbon of the substrate side chain (36, 37). Perhaps because of
this bias, it is rare to find a conventional protein kinase that
phosphorylates any one protein exclusively on threonine, much less a
whole set of proteins. To take one example, the RLC, which is
phosphorylated by A-CAT and C-CAT primarily on threonine residues, is a
substrate for multiple conventional protein kinases, including the
Ca2+-calmodulin-dependent protein kinase II,
cAMP-dependent kinase, casein kinase I, mitogen-activated
protein kinase-activated protein kinase-2, myosin light chain kinase,
PAK, phosphorylase kinase, protein kinase C, Rho kinase, and ZIP kinase
(38-45). All of these kinases, except casein kinase II,
phosphorylates the RLC either on serine or on serine and threonine
residues. Casein kinase II phosphorylates the RLC primarily on
threonine (39), but phosphorylates many other substrates, including
caldesmon, primarily on serine (46). We are unaware of any conventional
serine/threonine protein kinase that exhibits a strong and consistent
preference for the phosphorylation of threonine residues. The
preference displayed by the MHCK A type kinases for the bulkier
threonine side chain, together with the lack of sequence homology to
conventional protein kinases, suggests that the architecture of their
catalytic domain may be quite different from that of conventional
protein kinases. Attempts are presently underway to produce crystals of
A-CAT and C-CAT suitable for x-ray crystallographic analysis.
-helical
coiled-coil myosin II tail and that the eEF-2 kinase phosphorylates
sites located within an
-helix in eEF-2, it has been suggested these
kinases are specialized to recognize phosphorylation sites located
within
-helices (14). As a consequence, the name
-kinases has
been proposed for this kinase family (14). However, the results
reported here support the view that A-CAT, B-CAT, and C-CAT are capable
of phosphorylating short synthetic peptides and proteins that have
little or no
-helical structure in solution. MBP is a good
substrate for all three catalytic domains, yet secondary structure
prediction methods, circular dichroism data, and electron microscopic
three-dimensional reconstructions indicate that it has very low
-helical content (49-51). Casein proteins also have little
-helical content and a low degree of structural organization in
aqueous neutral solvents (52, 53), yet are substrates for A-CAT, B-CAT,
and C-CAT. Moreover, the ability to MHCK A and MHCK B to efficiently
phosphorylate the
-helical myosin II tail depends to a large extent
on the targeting function of the WD-repeat domain and does not seem to
be an intrinsic property of the catalytic domain (27).
4
and
2 positions and for basic residues in the +3 and +4 positions,
but differ in their selectivity for residues in the
1 and +1
positions (Table II). As a test of the validity of these results, a
synthetic peptide (YAYDTRYRR) corresponding to the predicted A-CAT
consensus sequence was synthesized and assayed for its ability to
function as an A-CAT substrate (Fig. 6A). Kinetic analysis
showed that A-CAT phosphorylated YAYDTRYRR with a specificity constant
(kcat/Km) of 0.025 µM
1 s
1, which is 4-fold
higher than the specificity constant for MH-3 (0.006 µM
1 s
1). By this criteria,
YAYDTRYRR is the best A-CAT peptide substrate yet identified.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. W. F. Loomis, University of California at San Diego, for providing DNA encoding MHCK C and M. Carpenter, Alberta Peptide Institute, for expert amino acid analyses and peptide sequence analyses.
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FOOTNOTES |
---|
* This work was supported in part by Canadian Institutes of Health Research Grant MOP8603 (to G. P. C.) and National Institutes of Health Grant GM50009 (to T. T. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an American Cancer Society Post-doctoral Fellowship.
¶ To whom all correspondence should be addressed: Dept. of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2998; Fax: 613-533-2497; E-mail: coteg@post.queensu.ca.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M009366200
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ABBREVIATIONS |
---|
The abbreviations used are: MHCK, myosin II heavy chain kinase; A-CAT, catalytic domain of MHCK A; B-CAT, catalytic domain of MHCK B; C-CAT, catalytic domain of MHCK C; eEF-2, eukaryotic elongation factor-2; GST, glutathione S-transferase; MBP, myelin basic protein; PAK, p21-activated kinase; RLC, myosin regulatory light chain; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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