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Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 248-251, January 1978 Biochemistry
In vivo phosphorylation of a synthetic^ peptide^ substrate^ of^ cyclic
AMP-dependent protein kinase
(Xenopus oocytes/microinjection/protein phosphorylation/peptides)
JAMES L. MALLER, BRUCE E. KEMPt,^ AND^ EDWIN G.^ KREBS
Department of Biological Chemistry, University^ of^ California,^ Davis,^ California^95616
Contributed by Edwin G. Krebs, November^ 4, 1977
ABSTRACT A^ model^ synthetic peptide^ substrate of the cyclic AMP-dependent protein kinase^ (ATP:protein^ phospho- transferase; EC 2.7.1.37), Leu-Arg-Arg-Ala-Ser-Leu-Gly,^ closely resembling the local^ phosphorylation^ site^ sequence^ in porcine hepatic pyruvate kinase, was^ shown^ to^ be^ phosphorylated^ in vivo after microinjection into^ Xenopus^ oocytes.^ This^ result demonstrates that the microinjection^ technique, utilizing^ a synthetic peptide substrate, or possibly a^ synthetic substrate analog inhibitor [Kemp, B. E., Benjamini, E.^ &^ Krebs,^ E.^ G.
(1976) Proc. NatL Acad. Sci. USA 73,1038-10421, can^ be used^ to
study protein phosphorylation-dephosphorylation reactions^ in
living oocytes. This follows, since it is clear that the^ injected peptide was accessible to the cellular compartment containing the protein kinase.
Since the^ discovery of^ cyclic AMP^ by^ Sutherland and Rall^ (1),
considerable advances have been made in understanding the
mechanism by which^ this^ important second^ messenger^ exerts
its effects on intracellular processes. It was established that^ a
cyclic AMP-dependent protein kinase^ (ATP:protein phospho-
transferase; EC^ 2.7.1.37)^ is^ the^ primary^ target^ for control^ of
glycogenolysis by cyclic AMP^ in^ skeletal^ muscle^ (2, 3)^ and^ the
generality of^ this mechanism^ as^ applied^ to^ other^ cyclic^ AMP-
stimulated systems was proposed (4). A^ considerable^ number
of natural substrates^ of the^ cyclic^ AMP-dependent^ protein^ ki-
nase have been identified, and their^ number^ and^ diversity of
function illustrate the important role^ this^ protein^ kinase^ plays
in coordinating various intracellular processes with^ the^ demands
of the organism. It is^ apparent, therefore, that^ the^ protein
substrate specificity of the protein kinase should have^ a^ crucial
role in determining which^ processes are^ modulated^ in^ response
to a given signal.
Recently, the molecular basis of the^ substrate^ specificity^ of
the cyclic AMP-dependent protein kinase^ has^ been studied^ by
using low molecular weight peptides (5-11). Studies^ with^ syn-
thetic peptides both in this laboratory and elsewhere^ (11) in-
dicate that the enzyme recognizes a^ rather^ restricted^ region^ of
the primary structure around the phosphorylation site^ (6-11).
The synthetic peptide substrates^ that^ have^ been^ most exten-
sively studied correspond to^ the^ phosphorylation site^ sequence
reported for pig^ liver^ and^ rat^ liver^ pyruvate^ kinase^ (10, 11).^ A
heptapeptide studied^ in^ this^ laboratory,^ Leu-Arg-Arg-Ala-
Ser-Leu-Gly, has an apparent Km in^ the low^ micromolar^ range
(10) and^ the^ Vnax with^ this^ substrate^ is^ of^ the^ same^ order^ as^ that
for physiological substrates.
Since the kinetic constants obtained^ with the^ heptapeptide
as substrate^ were^ of^ the^ same^ order^ as^ those^ obtained^ with
proteins as substrates, it seemed possible that^ this^ peptide would
be capable of competing with natural substrates^ in^ vivo^ if^ it
could cross^ the^ cell membrane.^ A^ suitable^ system^ for^ testing^ this
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248
is the living Xenopus oocyte into which^ volumes^ of up to^ 0.
,l can be injected with a micropipet. The question of phos-
phorylation of the peptide is also^ of^ interest^ since we have^ found
that meiosis in the oocyte, triggered by progesterone, is inhib-
ited by microinjection of micromolar levels^ of the^ homogeneous
catalytic subunit of the protein kinase and that it is induced
without hormone by microinjection of protein kinase^ inhibitory
proteins (12). These findings suggest that one of the substrates
for the catalytic subunit is necessary and sufficient in its phos-
phorylated form^ to^ maintain^ the^ prophase block^ of the^ oocyte
either directly or indirectly (12). If synthetic peptides at low
concentrations in^ vivo could act as^ competitive inhibitors^ of the
enzyme for normal substrates, as occurs in vitro (6), it might
be possible to alter the progress of^ meiosis^ by microinjection^ of
specific peptide^ substrates^ or^ suitable analogs.^ As^ a^ first step
towards determining the feasibility of this approach, we show
here that synthetic peptides introduced into oocytes of Xenopus
laevis can be phosphorylated, thus demonstrating^ that such
compounds, when injected, are accessible to the protein ki-
nase.
MATERIALS AND METHODS
Cyclic AMP-Dependent^ Protein^ Kinase.^ Homogeneous
beef skeletal muscle catalytic subunit was prepared by the
method of^ Beavo et^ al.^ (13).^ The^ catalytic^ subunit^ of cyclic
AMP-dependent protein kinase from Xenopus eggs was pre-
pared as described^ (12). Protein^ kinase^ activity^ was^ measured
by the method of Reimann et al. (14), with mixed histone type
II-A as^ substrate. When synthetic^ peptide^ was^ used^ as^ substrate,
aliquots of the reaction mixtures containing synthetic peptide
were applied to discs of Whatman-P81 paper and washed with
30% acetic acid (four times for 20 min each). The paper discs
were dried and radioactivity was determined in a Nuclear
Chicago scintillation counter with^ a toluene-based scintillant.
This ion-exchange filter paper procedure gives results quanti-
tatively similar to those of the anion-exchange column^ proce-
dure described previously (6). [,y-32P]ATP was prepared ac-
cording to^ the method of^ Glynn and^ Chappel^ (15).
Peptide Synthesis and Purification. The synthetic peptide,
Leu-Arg-Arg-Ala-Ser-Leu-Gly, was^ synthesized^ by^ the^ Mer-
rifield solid phase synthesis technique and^ purified^ by^ ion-
exchange chromatography on SP-Sephadex and gel chroma-
tography on^ Sephadex^ G-25^ (6).^ The^ amino^ acid^ composition
of the synthetic peptide after acid^ hydrolysis (5.7 M^ HCl, 1100,
24 hr) determined on a Durrum D-500 amino acid analyzer was
Ala (1.00), Arg (1.98), Gly (0.99), Leu (2.03), and Ser (1.00). The
quantitative yield after^ total^ enzymic hydrolysis with^ amino-
- (^) Present address: Department of Pharmacology, University of Washington, Seattle, WA 98195. t (^) Present address: Clinical Biochemistry Unit, The Flinders University of South Australia, Bedford Park, 5042, South Australia.
peptidase-M compared with acid hydrolysis was 101%, shwibg
that the synthetic peptide was fully deprotected and had
maintained its stereospecificity throughout the synthesis.
Microinjection of Qocytes. Ovaries were obtained from
healthy Xenopus laevis, and the oocytes collected by colla-
genase treatment (16). Only unblemished large 1.3-mm-di-
ameter (stage VI) oocytes were used.^ Microinjection procedures
together with the construction and calibration of micropipets
are described in detail elsewhere (17). Individual oocytes were
injected with (^) 32P, (2.5-3.5 (^) ,Ci per oocyte) and subsequently
with synthetic peptide (1.9-2.6 nmol) in a^ total volume of^ 50-
nl and were then incubated in Wallace's medium OR2 (18)
minus KC1 (19) and NaH2PO4, for^10 min.
Isolation of Phosphorylated Peptide. Oocytes with 32P1,
with or without synthetic peptide, were^ fixed^ in 3^ ml of^ 30%
acetic acid for 12 hr at 0°. This procedure left the oocytes
largely intact while allowing the synthetic peptide to^ leak^ out
into the fluid above the oocytes. In control experiments with
this technique, the recovery of phosphorylated peptide from
oocytes injected with 32P1-labeled phosphopeptide was greater
than 80%. This procedure was used after it was^ found that ex-
traction of the peptide from oocytes by oocytes by homogeni-
zation was complicated owing to^ the^ release^ of^ phosphorylated
components of the oocyte which were not readily separated
from the phosphorylated peptide. Phosphopeptide recovered
from the oocytes was separated from residual (^) 32P, and 32p-
labeled nucleotides by anion-exchange chromatography (AG
1X8 resin, Bio-Rad) in the presence of 30% acetic acid. For
isolation of peptide phosphorylated in^ vitro, reaction^ mixtures
(13) were diluted in 30% acetic acid and subjected to the same
chromatographic procedure.
RESULTS
Phosphorylation of the synthetic peptide, Leu-Arg-Arg-
Ala-Ser-Leu-Gly, in vivo
When oocytes were injected with carrier-free 32Pi plus synthetic
peptide and incubated for 10 min, approximately 109 X^103
cpm of^ 32P, per oocyte was^ recovered in^ the phosphopeptide
fraction after anion-exchange chromatography. In contrast,
only 4.4 X 103 cpm per oocyte was recovered in this fraction
from control oocytes injected with (^) 32P, without synthetic pep- tide.
The phosphorylated product isolated from the oocytes in-
jected with^ peptide was characterized as follows. By high-
voltage electrophoresis, it migrated toward the cathode in the
same position as authentic phosphopeptide that had been pre-
pared in vitro with the catalyted subunit of cyclic AMP-de-
pendent protein kinase isolated from either beef skeletal muscle
or Xenopus eggs (Fig. 1). As can be seen, several minor labeled
components from the in vivo reaction were also present, which
may represent partial proteolysis products derived from the
peptide. In extracts of control oocytes that had been injected
only with 32P1, there was no detectable 32P^ radioactive material
migrating towards the cathode. The radioactivity associated
with the peptide, phosphorylated either in vitro or in the intact
oocyte, was^ alkali-labile (94% released in^ 15 min in 0.1 M NaOH
at 100°) and stable to acid (1000 remaining after 15 min in 0.
M HC1 at 100°). These properties are consistent with the
presence of a serine phosphoester linkage in the phosphopeptide
isolated from the oocyte. When the phosphorylated product
was subjected to partial acid hydrolysis and high-voltage paper
electrophoresis at pH 1.9, radioactivity in the same position as
a phosphoserine marker was observed (Fig. 2).
The phosphorylated product obtained from the oocytes was
further characterized by thin-layer chromatography on silica
FIG. 1. Autoradiograph of an electrophoretogram of synthetic peptide phosphorylated in vivo and in vitro. The phosphopeptide fractions were concentrated by rotary evaporation and aliquots were electrophoresed for 2 hr at 1600 V (pH 4.7, 5% butanol/2.5% pyri- dine/2.5% acetic acid/water, by volume). Twenty-four-hour autora- diographs of the ninhy--in-stained electrophoretograms were pre- pared with Kodak x-ray film. E, synthetic peptide phosphorylated in (^) vivo after microinjection into oocytes; S, synthetic peptide phos- phorylated in^ vitro with catalytic subunit of beef skeletal muscle protein kinase; F, synthetic peptide phosphorylated in vitro with partially purified catalytic subunit of egg protein kinase; C, control oocytes microinjected with 32p, but without synthetic peptide.
gel plates with^ four different solvent systems (Fig. 3). In each case, the phosphorylated product isolated from the oocytes migrated with the same RF value as the phosphopeptide pre- pared in vdtro. A certain amount of streaking occurred on the thin-layer plates, particularly in the case of phosphopeptide prepared in vitro using the egg catalytic subunit (see Fig. 3). This may have been due to slight contamination of the partially purified enzyme preparation with proteases.
DISCUSSION The results reported here demonstrate that a model synthetic peptide substrate of the cyclic AMP-dependent protein kinase
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Biochemistry: Maller^ et^ al.
because it is phosphorylated in^ vitro^ by^ the^ catalytic-subunit
of the presumably analogous enzyme from eggs. It^ is^ possible,
of course, that after microinjection other^ protein^ kinases^ could
also phosphorylate the peptide substrate to yield an electro-
phoretically identical^ product.^ Hence,^ it^ cannot^ be^ concluded
with absolute certainty what enzyme was acting in^ this^ case.
For example, cyclic^ GMP-dependent^ protein^ kinase^ has^ a
substrate specificity similar to that of cyclic AMP-dependent
protein kinase and can phosphorylate in^ vitro^ the^ peptide^ used
in this study (29). Interestingly, the biological activity inside
the oocyte of cyclic GMP-dependent protein kinase^ kinase^ also
parallels the cyclic AMP-dependent enzyme, since the homo-
geneous cyclic GMP-dependent enzyme also^ inhibits^ meiosis
when microinjected, half-maximal inhibition occurring at an
internal concentration of approximately 50 nM.t
The most important specificity determinant for substrates
of cyclic AMP-dependent protein kinase appears to be^ the
presence of arginine residues^ near^ the^ phosphorylatable^ serine.
These particular residues also serve as determinants for^ tryp-
sin-like proteases, and thus^ it is^ not^ surprising^ that^ the^ peptide
substrate used in this study was susceptible to partial degrada- tion after microinjection (Fig. 1). This finding^ demonstrates that protein kinases and proteases share or overlap the same intra- cellular compartment and suggests experiments to^ compare^ the
susceptibility of nuclear and cytoplasmic proteins or peptides
or proteolytic degradation as a function of the^ region of^ the
oocyte into^ which^ they are^ introduced^ by^ micropipet. This^ kind
of approach might prove useful in^ characterizing the^ reported
involvement of proteases in meiotic maturation of Xenopus
oocytes (30).
Purified protein kinase catalytic subunit^ was^ generously supplied by Dr. Peter J. Bechtel, and Ms. Edwina Beckman kindly undertook the amino acid analysis. We are grateful to Dr. T. M. Lincoln of Van- derbilt University and Dr. David Glass of this laboratory for a gift of homogeneous cyclic GMP-dependent protein kinase. We thank Prof. J. Hedrick^ for^ providing^ healthy^ Xenopus^ laevs.^ B.E.K.^ is^ a^ recipient of an Australian National Heart Foundation Overseas Fellowship. J.M. is (^) the recipient of a postdoctoral fellowship from the Muscular Dys- trophy Association of America. E.G.K. is an Investigator of the Howard Hughes Medical Institute.^ This^ work^ was^ supported^ by^ grants^ from^ the National Institutes of Health (AM 12842 and AM 16716).
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Biochemistry: Maller^ et^ al.
