(Received for publication, March 30, 1995; and in revised form, June 27, 1995)
From the
The S6/H4 kinase purified from human placenta catalyzes phosphorylation of the S6 ribosomal protein, histone H4, and myelin basic protein. In vitro activation of the p60 S6/H4 kinase requires removal of an autoinhibitory domain by mild trypsin digestion and autophosphorylation of the catalytic domain (p40 S6/H4 kinase). The two autophosphorylation/autoactivation sites contain the sequences SSMVGTPY (site 1) and SVIDPVPAPVGDSHVDGAAK (site 2). These sequences identify S6/H4 kinase as the rac-activated PAK65 (Martin, G. A., Bollag, G., McCormick, F. and Abo, A.(1995) EMBO J. 14, 1971-1978). Site 1 phosphorylation is most rapid, but activation does not occur until site 2 is autophosphorylated. The site 1 phosphorylation occurs by an intramolecular mechanism whereas site 2 autophosphorylation occurs by an intermolecular mechanism. A model is proposed in which phosphorylation of sites 1 and 2 occurs sequentially. The model proposes that trypsin treatment of the inactive holoenzyme removes an inhibitory rac-binding domain which blocks MgATP access to the catalytic site. The pseudosubstrate domain at site 1 is autophosphorylated and subsequent bimolecular autophosphorylation at site 2 fully opens the catalytic site. Phosphorylation by a regulatory protein kinase may occur at site 2 in vivo.
Cellular activation of serine/threonine protein kinases occurs as a result of conformational changes induced by binding of small ligands, such as cyclic AMP or calcium, binding of regulatory proteins, such as Ras, Rho, or cyclin, or phosphorylation by upstream protein kinases (reviewed in Refs. 1, 2). Although the events which trigger activation of serine/threonine protein kinases are quite variable, a common molecular mechanism by which the enzyme inhibition is relieved has been proposed(3) . Intrasteric activation, the conformational changes at the active site which result in increased accessibility to MgATP, protein substrate, or both, has been proposed as a common feature of protein kinase activation. Data obtained by x-ray crystallography demonstrate that in the inactive enzyme conformation, auto-inhibitory or pseudosubstrate domains of the protein kinase bind at the protein substrate-binding site and restrict access of substrates to the catalytic center(4, 5, 6, 7, 8) . As demonstrated in studies of both the cyclic AMP-dependent protein kinase and the twitchin kinase, the autoinhibitory or pseudosubstrate domain competes effectively with exogenous protein substrates for enzyme binding and may also block binding of MgATP in the catalytic cleft(4, 5, 9) . Displacement of the autoinhibitory domain from the active site occurs when conformational changes initiated by the regulatory ligand binding decrease the binding affinity of the pseudosubstrate domain.
In some protein kinases,
phosphorylation of the autoinhibitory domain may be required for
displacement from the catalytic center and subsequent activation. Most
protein kinases catalyze autophosphorylation although for many
serine/threonine protein kinases, this self-modification does not alter
catalytic function. However, recently several protein kinases which
require autophosphorylation for activation have been
reported(10, 11, 12, 13, 14) .
In this laboratory we have demonstrated that the catalytic domain of an
S6/H4 kinase ()from human placenta undergoes rapid
autophosphorylation concomitant with activation, and inhibition of
autophosphorylation is correlated with inhibition of phosphotransferase
activity(10) . Similar results have been observed in studies of
a pig liver glycogen synthase kinase(11) , a bovine kidney
phosphatase 2A kinase(12) , an interferon-induced,
RNA-dependent protein kinase(13) , and a rac-activated brain
enzyme which has sequence identity to the STE20 yeast gene
product involved in pheromone signaling(14) . The diversity of
these enzymes with respect to physical and enzymatic properties
suggests that a requirement for autophosphorylation may be a widespread
occurrence within the serine/threonine protein kinase family.
In this report we have investigated the mechanism by which autophosphorylation of the S6/H4 kinase occurs. By analogy to the pseudosubstrate domain hypothesis, we propose that a regulatory domain in the S6/H4 kinase binds at the protein substrate-binding site. However, the data suggest that in the S6/H4 kinase, this domain contains a serine which must become phosphorylated in order for it to be displaced from the catalytic cleft and permit binding of exogenous substrates. In addition, maximum activation of the S6/H4 kinase appears to require a second phosphorylation on the surface of the enzyme.
For autophosphorylation studies, the reaction was
stopped after the MgATP incubation by the addition of SDS sample
buffer, and the reactions products were analyzed by
SDS-PAGE(18) . The resolved proteins were transferred to
nitrocellulose membranes in Towbin buffer (19) and the
radiolabeled bands detected by autoradiography. Radioactivity was
quantitated by excising the bands from the membrane and counting the PO
3 incorporation by the
Cerenkov method or in 10 ml of toluene-based scintillation fluor.
For sequencing experiments, S6/H4 kinase (1 mg/ml) was incubated with 5% (w/w) diphenylcarbamyl chloride-treated trypsin in 0.1 M ammonium bicarbonate, pH 7.8, for 24 h at 37 °C. Half of the trypsin was present for the first 12 incubations; the remaining trypsin was added after 12 and 18 h of incubation.
Figure 1:
Two- dimensional peptide map of p40
S6/H4 kinase autophosphorylation sites. S6/H4 kinase (4.8 µg/100
µl) was incubated with trypsin for 5 min in the presence of 8 mg/ml
myoglobin. The trypsin reaction was stopped with soybean trypsin
inhibitor, and the reaction mixture was incubated for 10 min with
Mg[-
P]ATP (4500 dpm/pmol). The
phosphorylation reaction was stopped by the addition of SDS-PAGE sample
buffer, and the proteins were analyzed by 10% PAGE. Protein was
transferred to nitrocellulose, and the
P-labeled p40
protein was trypsin digested as described under ``Experimental
Procedures.'' The tryptic peptides were applied to a cellulose
plate and TLE at pH 1.9 and TLC carried out as described under
``Experimental Procedures.'' Radioactive peptides were
detected by autoradiography (shown). Phosphopeptides are numbered from
slowest to fastest migration toward the
cathode.
To determine that none of the observed peptides
were generated by incomplete digestion with trypsin or by the
occurrence of multiple oxidation states of a single peptide,
[P]p40 S6/H4 kinase immobilized on
nitrocellulose was digested with trypsin for times ranging from 2 to 6
h, and the tryptic peptides were subjected to performic acid/hydrogen
peroxide oxidation, as described under ``Experimental
Procedures.'' Neither of these treatments changed either the
number of phosphopeptides observed on the TLE/TLC maps or the relative
amounts of the peptides (data not shown).
The number of
autophosphorylation sites in purified p40 S6/H4 kinase was also
investigated. Peptides obtained from trypsin digestion of
[P]p40 S6/H4 kinase were analyzed by HPLC. In
agreement with phosphopeptide maps obtained from in situ trypsin treatment and autophosphorylation (Fig. 1), four
radioactive peptides were detected in the column eluate (Fig. 2). Each phosphopeptide peak was pooled individually and
analyzed by TLE at pH 1.9. Each of the four peaks resolved by HPLC
corresponded to one of the phosphopeptides observed in Fig. 1,
and the peaks in Fig. 2are numbered according to the
corresponding phosphopeptide in the TLE/TLC map. In all of the HPLC
analysis of fully phosphorylated [
P]p40 S6/H4
kinase phosphopeptides, phosphopeptide 2 was most abundant and
phosphopeptide 4 was least abundant and was barely detectable in some
experiments. The relative amounts of phosphopeptides 1 and 3 were more
variable. In most experiments, phosphopeptide 1 was the second most
abundant phosphopeptide observed, being 50-60% of phosphopeptide
2 at maximum activation. Phosphopeptide 3 occurred in half or less the
amount of phosphopeptide 1. However, in approximately one-third of the
experiments, the amount of phosphopeptide 3 was nearly equal that of
phosphopeptide 1. When phosphopeptides obtained from trypsin digestions
of immobilized [
P]p40 S6/H4 kinase were compared
to results from [
P]p40 S6/H4 kinase digested in
solution, the same phosphopeptides in the same relative concentrations
were observed (data not shown).
Figure 2:
HPLC analysis of p40 S6/H4 kinase
autophosphorylation sites. Purified p40 S6/H4 kinase (16.2 µg) was
incubated for 20 min with Mg[-
P]ATP (7500
dpm/pmol), analyzed by 10% PAGE, transferred to nitrocellulose, and the
P-labeled p40 protein was digested with trypsin in
situ as described under ``Experimental Procedures.'' The
tryptic peptides were applied to a reverse phase C
column
and eluted in 1-ml fractions at 1 ml/min with a linear gradient of
2.5-50% acetonitrile as described under ``Experimental
Procedures.'' The A
(upper panel)
and
P-labeled peptides (lower panel) were
determined. The phosphopeptides are labeled according to their R
values with TLE as shown in Fig. 1.
The amino acid sequences of purified phosphopeptides 1 and 2 were determined. For phosphopeptide 1 the partial sequence SSMVGTPY was obtained. The failure to detect a lysine or arginine residue at the carboxyl terminus indicates that this sequence is most likely an incomplete sequence for phosphopeptide 1. The sequence SVIDPVPAPVGDSHVDGAAK was obtained for phosphopeptide 2. It appears likely that this is the complete sequence for this peptide. These data establish that there are at least two distinct phosphorylation sites in p40 S6/H4 kinase.
Since serine, threonine, and tyrosine residues occur in the phosphopeptide sequences, the identity of the phosphoamino acid in p40 S6/H4 kinase was determined. Only phosphoserine was detected (Fig. 3). The failure to detect phosphothreonine or phosphotyrosine and the observation that dehydroalanine was detected in the first rounds of phosphopeptides 1 and 2 sequencing supports the conclusion that the serine residues at the beginning of both peptides are the phosphorylation sites, and neither the threonine nor the tyrosine residues are autophosphorylated during the in vitro autophosphorylation and activation.
Figure 3: Phosphoamino acid analysis of autophosphorylated p40 S6/H4 kinase. Autophosphorylated p40 S6/H4 kinase was prepared as described in Fig. 2. The protein was partially hydrolyzed with 6 N HCl at 110 °C for 1 h. An aliquot of the hydrolysate was applied to a microcyrstalline cellulose plate and TLE conducted at pH 1.9 (bottom to top) and pH 3.5 (left to right). Phosphoamino acids were visualized by autoradiography (shown) and identified by comparison to phosphoamino acid standards as described under ``Experimental Procedures.'' The location of the standards is indicated.
Figure 4:
Effect of p40 S6/H4 kinase concentration
on the time course for activation with MgATP. Purified p40 S6/H4 kinase (closed circles, 96 ng/µl; open circles, 76
ng/µl; closed squares, 48 ng/µl) was incubated with
Mg[-
P]ATP (230 dpm/pmol) for the designated
time intervals. After the activation time was completed, S6-21
(190 µM) was added to the reaction and the protein kinase
assay was carried out for 10 min.
The lag time for activation was well correlated with a lag time for phosphorylation of site 2, but not site 1 (Fig. 5). At a concentration of p40 S6/H4 kinase which was activated less than 15% after 5 min of incubation with MgATP, phosphorylation of site 2 was barely detectable whereas phosphorylation of sites 1, 3, and 4 were nearly maximum. After 10 min of incubation with MgATP, both activation and autophosphorylation of site 2 were approximately 60% of maximum.
Figure 5:
Correlation of site-specific p40 S6/H4
kinase autophosphorylation with activation time. Purified p40 S6/H4
kinase (42 ng/µl) was incubated with
Mg[-
P]ATP (7000 dpm/pmol) for 3, 5, or 10
min (lanes A-C, respectively). Phosphopeptides from trypsin
digests were prepared as described in Fig. 2, and the peptides
were analyzed by TLE at pH 1.9 followed by autoradiography (shown). The
phosphopeptides were compared quantitatively by computer scanning the
autoradiograph and activation was determined as described in Fig. 4(not shown).
The activation kinetics of p40 S6/H4 kinase were determined by incubating various dilutions of p40 S6/H4 kinase with MgATP for 2, 4, or 10 min prior to assay with H4 as the protein substrate (Fig. 6). At all three activation times, no direct proportionality between enzyme activity and enzyme dilution was observed. In control experiments in which activation was carried out for 10 min and the time course for the assay with H4 was monitored, linear incorporation of phosphate into H4 was observed, demonstrating that the nonlinearity of activity with respect to dilution reflected cooperativity in the activation kinetics, and not in the H4 assay (data not shown).
Figure 6:
Effect of p40 S6/H4 kinase concentration
on the kinetics of activation with MgATP. Varying concentrations of
purified p40 S6/H4 kinase were incubated with
Mg[-
P]ATP for 10 min (closed
circles), 4 min (open circles), or 2 min (closed
squares). After activation, H4 (1 mg/ml) was added and all
reaction mixtures were assayed for 10 min.
When the enzyme concentration was increased 2-fold from 10.3 to 20.6 ng/µl, and the enzyme was activated for 2 min with MgATP, the activity of the enzyme with the H4 substrate increased 4.2-fold from 1.9 to 7.9 pmol/min. Activation of the same enzyme concentrations for 4 min resulted in a 5.5-fold activity change from 4.8 to 26 pmol/min. Linear regression analysis of the data in Fig. 6indicated that for all of the MgATP activation times studied, the change in enzyme activity with respect to enzyme dilution was better fit to a curve than to a straight line. For the 10-min activation time, the correlation coefficient was 0.993 for the curved line but 0.866 for a straight line. Since the data fit the curved line plot better, activation of the S6/H4 kinase by an intermolecular autophosphorylation is suggested. These data do not eliminate the possibility that an intramolecular autophosphorylation is also involved in the activation mechanism.
Figure 7:
Effect of p40 S6/H4 kinase concentration
on the kinetics of specific site autophosphorylation. p40 S6/H4 kinase
was incubated with Mg[-
P]ATP (25,000
dpm/pmol) for 10 min. Phosphopeptides were generated and analyzed by
HPLC as described in Fig. 2. Total radiolabel incorporated into
site 2 (upper panel) or site 1 (lower panel, solid circles), site 3 (lower panel, closed
squares), or site 4 (lower panel, open squares)
was determined by liquid scintillation counting of the HPLC
fractions.
Site 1 phosphorylation was linear with respect to enzyme dilution, suggesting that this modification occurs intramolecularly (Fig. 7). In this experiment, phosphorylation of phosphopeptide 3 was too low to determine a mechanism reliably. However, the relative amount of phosphopeptide 1 and phosphopeptide 3 label varied somewhat from experiment to experiment, and other data indicate that site 3 autophosphorylation exhibits linear kinetics (data not shown). The stoichiometry of site 4 phosphorylation was so low in most experiments that no firm conclusion regarding its kinetics could be drawn. These data suggest that site 1 and 3 autophosphorylation are intramolecular reactions, with site 1 being the major modification and site 3 a minor modification and that both of these phosphorylations occur with no demonstratable time lag.
Figure 8:
Intermolecular phosphorylation and
activation of S6/H4 kinase by p40 S6/H4 kinase. p40 S6/H4 kinase (1.4
µg, lanes A and C) or S6/H4 kinase (19.4 µg, lane B) was incubated with
Mg[-
P]ATP (4500 dpm/pmol) for 10 min. The
incubation was stopped by the addition of SDS-PAGE sample buffer (lanes A and lane B) or continued for 10 min with the
addition of S6/H4 kinase (19.4 µg) to the p40 S6/H4 kinase (lane C). After the total incubation time, samples were
treated with SDS-PAGE sample buffer, analyzed by SDS-PAGE, and
transferred to nitrocellulose. Phosphoproteins were detected by
autoradiography (shown).
p40 S6/H4 kinase was autophosphorylated after the 10-min incubation with MgATP, and this autophosphorylation was increased when the MgATP incubation time was extended to 20 min (Fig. 8, lanes A and C). Little autophosphorylation was detected when p60 S6/H4 kinase alone was incubated with MgATP (Fig. 8, lane B). However, when p60 S6/H4 kinase was incubated with p40 S6/H4 kinase (Fig. 8, lane C), an increase in p60 phosphorylation was detected.
When phosphopeptides from the immobilized p60 bands were analyzed by trypsin digestion and HPLC analysis, an increase in site 2 phosphorylation, but not in site 1 or 3 phosphorylation, was observed (data not shown). In control experiments where an equivalent amount of holoenzyme was activated by trypsin and MgATP, sites 1 and 2 were both actively phosphorylated. These results confirm the hypothesis that site 2 phosphorylation occurs by a bimolecular mechanism and activated p40 S6/H4 kinase can catalyze this reaction.
To correlate p60 S6/H4 kinase site 2 phosphorylation with activation, the change in activity after phosphorylation by p40 S6/H4 kinase was determined. This experiment was designed so that as little as 5% activation of the p60 S6/H4 kinase holoenzyme could be detected in the presence of the catalytic amounts of p40 S6/H4 kinase used. In control samples, S6/H4 kinase (2.5 µg) and p40 S6/H4 kinase (0.5 µg) were activated fully by trypsin and MgATP incubation and assayed in the presence of H4. The combined total activity of the two enzymes was 12.8 pmol/min; the activity of p40 S6/H4 kinase alone incubated with MgATP for 10 min was 1.1 pmol transferred/min. When p60 S6/H4 kinase was incubated with MgATP alone, the observed activity was 0.22 pmol transferred/min. The observed activity of p60 S6/H4 kinase preincubated with MgATP and p40 S6/H4 kinase was 1.5 pmol transferred/min; based on the amount of phosphate incorporated into site 2, an activity of 3.5 pmol/min would be predicted if site 2 alone was sufficient for activation. Since p60 S6/H4 kinase was phosphorylated at site 2 by p40 S6/H4 kinase, and no increase in activity over that predicted for the p40 S6/H4 kinase alone was detected, these data indicate that site 2 phosphorylation of p60 S6/H4 kinase is necessary, but not sufficient to activate the holoenzyme.
The results from the previous experiment do not exclude
the possibility that site 2 autophosphorylation is sufficient to
activate the catalytic domain (p40 S6/H4 kinase) of the enzyme. To test
if site 2 phosphorylation was sufficient to activate p40 S6/H4 kinase,
p60 S6/H4 kinase was phosphorylated with p40 S6/H4 kinase as described
above, and the unreacted MgATP was removed by passing the enzyme
mixture over a superfine G25 column (1.5 ml) equilibrated in 0.1 M Tris-Cl, pH 7.5, containing 2 mM EDTA, 2 mM EGTA, 10 mM 2-mercaptoethanol, 2 µM leupeptin, 0.l mM phenylmethylsulfonyl fluoride, 10%
glycerol, and 0.2 M NaCl. Protein-containing fractions were
detected by spot Bradford analysis, and
Mg[-
P]ATP-containing fractions were
detected by liquid scintillation counting. The peak protein-containing
fraction which did not overlap the
Mg[
-
P]ATP elution was used to test for
activation of the enzyme by site 2 phosphorylation. To determine the
activity of p40 S6/H4kinase with site 2 phosphorylated, the S6/H4
kinase obtained from the Sephadex G-25 chromatography was assayed in
the presence and absence of trypsin and MgATP-dependent activation.
The p60 S6/H4 kinase which was phosphorylated by p40 S6/H4 kinase at site 2 could be fully activated by subsequent incubation with trypsin and MgATP, but no activation was observed in the absence of trypsin treatment either with or without incubation with MgATP (65 pmol phosphate transferred/min versus 3.2 pmol phosphate transferred/min). When the sample was treated with trypsin, but no preincubation with MgATP, the observed activity was 29% of the total activity elicited by trypsin and MgATP treatment. This amount of activity did not differ from that observed when a comparable amount of p60 S6/H4 kinase and p40 S6/H4 kinase were treated with trypsin and assayed with H4 histone without activation by autophorylation. It is likely that amount of activation occurs in the assay as a result of incomplete inhibition of autophosphorylation by H4 in the presence of these relatively high concentrations of p40 S6/H4 kinase. These data indicate that site 2 phosphorylation is not sufficient to activate either p60 or p40 S6/H4 kinase and that intramolecular site 1 autophosphorylation is also required for enzyme activation.
Both
site 1 and 2 phosphorylation appear to be required for S6/H4 kinase
activation, but the data presented do not establish that both
modifications occur on the same enzyme molecule. To exclude the
possibility that site 1 and 2 phosphorylation were mutually exclusive,
phosphorylated p40 S6/H4 kinase was analyzed by two-dimensional
SDS-PAGE and isoelectric focusing (Fig. 9). Two prominent P-labeled enzyme forms were observed when samples
incubated with Mg[
-
P]ATP for 3 and 15 min
were subjected to isoelectric focusing in a pH 5-7 polyacrylamide
gel. At the shorter time, the amount of radiolabel in the two spots was
approximately equal. Since the more acidic form has at least two
phosphates, the data predict that approximately 66% of the
P-labeled enzyme is monophosphorylated at this time point.
After maximum activation by incubation for 15 min with
Mg[
-
P]ATP, the more acidic form, i.e. more highly phosphorylated form, predominated. These data
establish that after maximum phosphorylation, the major portion of the
enzyme contains two modified sites. After SDS-PAGE in the second
dimension, all enzyme isoforms migrated with M
40,000; the retardation of migration rate observed with many
phosphorylated proteins was not observed with the S6/H4 kinase.
Figure 9:
Resolution of p40 S6/H4 kinase
phosphoprotein isoforms by two-dimensional PAGE. Purified p40 S6/H4
kinase (0.3 µg) was incubated with
Mg[-
P]ATP (1700 dpm/pmol) for 3 min (top panel) or 15 min (bottom panel). The reaction
was stopped by the addition of isofocusing sample buffer containing 9 M urea. Samples were electrophoretically focused to their
isoelectric points in a pH gradient from 5 to 7 (horizontal direction).
The isofocusing gel was annealed to a 10% SDS-polyacrylamide gel and
the proteins resolved by molecular weight (vertical direction). The
phosphoproteins were detected by autoradiography (shown). Only the p40
portion of the SDS-PAGE is shown. The gel was silver stained to
determine the pI of nonphosphorylated p40 S6/H4 kinase (pH 6.3, not
shown).
Although the conserved domains of many protein kinases and the crystal structure of a small number of protein kinases suggest that this large enzyme family shares a common molecular organization in the catalytic core, the molecular mechanisms by which inhibitory domains are displaced from the catalytic cleft during activation may vary substantially. In the cyclic AMP-dependent protein kinase, the binding energy of the second messenger causes a conformational change in the regulatory subunit sufficient to break key bonds which anchor an inhibitory domain in the protein-binding groove of the catalytic site(4, 5) . In contrast, the mechanism by which the inhibitory domain of twitchin, a homologue of a myosin light chain kinase, blocks catalysis appears more global(3, 6) . In the case of this enzyme, the inhibitory domain sterically interferes with MgATP binding and blocks key catalytic residues in addition to binding in the protein substrate-binding groove. The activation of both MAP kinase and cdk requires phosphorylation by an exogenous protein kinase(2) . Crystal structures reveal that these modifications are not in the substrate-binding groove, but rather modulate the accessibility of key residues required for high affinity MgATP binding(7, 8) .
Activation of the S6/H4 kinase appears to require removal of an inhibitory domain which can be accomplished by mild trypsin digestion in vitro(10, 23) . Since the trypsin-activated enzyme quickly binds MgATP and autophosphorylates, the holoenzyme appears to be inhibited as the result of a blocked MgATP substrate-binding site. Removal of the 20-kDa trypsin-sensitive regulatory domain rac-binding domain (24) is not sufficient to permit productive binding of MgATP and protein substrate at the active site. Autophosphorylation is required to remove the pseudosubstrate domain from the active site and generate a fully active enzyme.
Two autophosphorylation reactions occur quickly after MgATP is added to the p40 S6/H4 kinase form of the enzyme. First, phosphorylation of the enzyme at site 1 occurs. Analysis of the kinetics of this reaction with enzyme dilution and the MgATP activation time course establish that this reaction occurs by an intramolecular mechanism and precedes activation of the enzyme. At MgATP incubation time intervals where no activation of the enzyme is detected, site 1 is phosphorylated to 30-60% of the maximum observed at full activation. However, this reaction appears to be essential for activation since neither p60 nor p40 S6/H4 kinase which is phosphorylated at only site 2 is active. Since the substrate-binding site is blocked in the nonactivated p40 S6/H4 kinase and since this reaction is intramolecular, a logical prediction is that this phosphorylation occurs at a pseudosubstrate inhibitory domain which is bound at the active site. After autophosphorylation, the phosphorylated pseudosubstrate domain likely behaves as reaction product and binds in the substrate-binding groove with less affinity. This model is supported by data published after submittal of this report(24) . Both site 1 and 2 sequences are predicted by the cDNA clone of a serine kinase which is activated by the GTP-binding protein rac and autophosphorylation. The site 1 sequence (amino acids 384-391) is adjacent to the APE sequence (amino acids 394-396) which positions the sequence in the catalytic cleft, consistent with the hypothesis that this is a pseudosubstrate domain.
The importance of the intermolecular phosphorylation at site 2 is more speculative. This modification is necessary, but not sufficient for activation. One possible role which might be predicted for this reaction is that the site 2 domain displaces the phosphorylated site 1 from the substrate-binding groove. If the autoinhibitory site 1 domain does not fully dissociate from the catalytic center after phosphorylation, such a mechanism might be required to attain the fully active conformation in which both MgATP and protein substrate bind with high affinity. This mechanism would predict that any exogenous substrate which binds with affinity high enough to compete with site 1 binding could fulfill the requirements for the second site phosphorylation in the activation mechanism. None of the current data exclude this possibility.
A second hypothesis for the role of site 2 phosphorylation addresses the in vivo mechanism for S6/H4 kinase activation. Although p40 S6/H4 kinase can catalyze phosphorylation of p60, this reaction is much slower than phosphorylation of substrates S6-21 and H4 by fully activated enzyme. This observation suggests that site 2 may be phosphorylated by a cellular kinase other than the S6/H4 kinase, i.e. an upstream regulatory kinase. In this case the role of the site 2 domain may be to alter the substrate-binding groove conformation sufficiently to facilitate the dissociation of phosphorylated site 1 from the catalytic site. In addition, activation by rac binding may promote a conformational change which alters the requirement for site 2 autophosphorylation.
Collectively, the data suggest that only sites 1 and 2 phosphorylations are required for activation of the trypsin-treated kinase. However, in some experiments phosphopeptide 3 is modified to nearly the same extent as phosphopeptide 1, and the kinetics of site 3 phosphorylation always parallel the kinetics of site 1 phosphorylation. In addition, the pI of p40 S6/H4 kinase is 6.3 whereas the putative mono- and diphosphorylated forms exhibited pI of 5.9 and 5.8, respectively. These data suggest that three or more phosphorylation reactions may occur. Since the amount of endogenous phosphorylation in each enzyme preparation may vary, a role for site 3 or site 4 cannot be completely excluded. Alternatively, site 3 may represent an alternate trypsin cleavage of site 1. Previous studies have demonstrated that the S6/H4 kinase requires a dibasic sequence, preferably K-R, amino-terminal to the modified serine(25) . Depending on the enzyme/trypsin ratios and extent of endogenous phosphorylation, site 3 may be generated by trypsin cleavage between the K and R, as opposed to cleavage at the carboxyl side of R for site 1. This hypothesis is consistent with the observation that phosphopeptide 3 exhibits TLC properties similar to phosphopeptide 1, but is more basic at neutral pH. This prediction would also be consistent with the observation that site 3 phosphorylation kinetics mimic site 1 phosphorylation kinetics. Consistent with the hyothesis that phosphopeptides 1 and 3 may reflect phosphorylation of the same serine, the predicted amino acid sequence (24) confirms that there are both a lysine and an arginine amino-terminal to the autophosphorylation site.