Table Of ContentTHE ENZYMES
Edited by
Paul D. Boyer Edwin G. Krebs
Department of Chemistry and Biochemistry Howard Hughes Medical Insiiiute
and Molecular Biology Insiitute and Department of Pharmacology
university of California University of Washington
Los Angeles. California Seattle, Washington
Volume XVII
CONTROL BY PHOSPHORYLATION
Part A
General Features
Specific Enzymes (I)
THIRD EDITION
1986
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The Enzymes.
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Preface
Over the past two decades there has been a remarkable increase in the recogni-
tion of the salient importance and the wondrous complexity of the control of
enzyme catalysis. The modulation of enzymic and other protein-dependent
processes by protein phosphorylation or dephosphorylation has emerged as the
most widespread and important control achieved by covalent modification. So
much information has emerged that adequate coverage in two volumes (XVII
and XVIII) was a challenging task. The editors are gratified that the contrib-
uting authors have commendably met this challenge.
The first portion of Volumes XVII and XVIII concerns the “machinery” of
control by protein phosphorylation and dephosphorylation and includes coverage
of the major types of protein kinases and of phosphoprotein phosphatases. The
central core of the volumes presents chapters on the control of specific enzymes.
This is followed by a substantial final section on the control of biological
processes.
The selection of authors for various chapters was a rewarding experience, but
made somewhat difficult because for most topics there was more than one well-
qualified potential author. The quality of the volumes was assured by the wel-
come acceptance of the invitation to participate by nearly all of the invited
authors.
The reversible covalent modification of enzymes and of proteins with other
functions is now known to occur in all types of cells and in virtually all cellular
compartments and organelles. Enzymes as a group constitute those proteins
whose function and control are best understood in molecular terms. The treat-
ment of enzymes gains additional importance because their regulation provides
prototypic examples to guide investigators studying less well defined and often
less abundant proteins. The versatility of protein control by phosphorylation
finds expression in ion channels, hormone receptors, protein synthesis, contrac-
tile processes, and brain function. Chapters in these areas point the way for
future exciting developments.
Although the breadth of coverage is in general regarded as satisfying, there are
other topics or areas that may have warranted inclusion. These include the
ix
X PREFACE
developing knowledge of the control by phosphorylation of histones of the nu-
cleus and the messenger-independent casein kinases, whose role is not as clear as
that of the major protein kinases that respond to regulatory agents.
The quality of the volumes has been crucially dependent on the editorial
assistance of Lyda Boyer and the fine cooperation provided by the staff of
Academic Press. We record our thanks here.
As readers of this Preface have likely discerned, it is a pleasure for the editors
to have volumes of high quality to present to the profession.
Paul D. Boyer
Edwin G. Krebs
The Enzymology of Control
by Phosphorylation
EDWIN G. KREBS
Howard Hughes Medical Institute and
Department of Pharmacology
University of Washington
Seattle, Washington 98195
1. Historical Aspects of Protein Phosphorylation ......................... 3
II. Protein Phosphorylation-Dephosphorylation Reactions 4
A. General Properties ..................................... 5
B. Specificity for Phosphoryl Donors in Protein Kinase Reactions 6
C. Protein Substrate Specificity of Protein Kinases and
Phosphoprotein Phosphatases .................... 6
D. The Reversibility of Protein K ... 8
E. Autophosphorylation Reactions ................................. 9
111. Classification of Protein Kinases and Phosphoprotein Phosphatases ....... 10
A. Protein Kinases ........................................ LO
B . Phosphoprotein Phosphatases ................................... 14
IV. Protein Phosphorylation and the Regulation of Biological Processes ....... 15
A. Phosphorylation-Dephosphorylation versus
Other Control Mechanisms ................... 15
B. Diversity of Phosphorylatio
Control Mechanisms .......................................... 17
References ........................................ 18
1. Historical Aspects of Protein Phosphorylation
Of the many types of posttranslational modification of proteins that occur in
cells relatively few are readily reversible. Those that are include acetylation,
3
THE ENZYMES, Vol. XVlI
Copyright 0 1986 by Academic Press. Inc.
All rights of repduction in any form reserved.
4 EDWIN G. KREBS
methylation, adenylylation, uridylylation, phosphorylation, and possibly one or
two others such as ADP ribosylation. By all odds the most common type of
reversible protein modification is phosphorylation, the process with which this
volume is concerned. As has become abundantly clear nature has chosen phos-
phorylation-dephosphorylation as an almost universal mechanism for regulating
the function of proteins, not only those that display enzymic activity but also
proteins involved in many other biological processes.
General recognition that protein phosphorylation has a major role in regulating
protein function developed over a protracted period of time rather than being
appreciated immediately as happened for allosteric regulation after the revela-
tions by Monod et af. (1) in the mid-1960s. The first dynamic protein phos-
phorylation-dephosphorylation system to be elucidated was that involving
glycogen phosphorylase, an enzyme that had been known to exist in two inter-
convertible forms, phosphorylases b and a (2, 3). In the mid-1950s these forms
were shown to be nonphosphorylated and phosphorylated species of the eyzyme,
which could be interconverted through the action of a protein kinase and a
phosphoprotein phosphatase (4, 5). In 1959 evidence was obtained that the
kinase involved in this process, phosphorylase kinase, was itself regulated by
phosphoxylation-dephosphorylation (6). A few years later it was determined that
glycogen synthase also exists in interconvertible phosphorylated and non-
phosphorylated forms (7).
The fact that the first three phosphorylatable enzymes to be discovered all
involved glycogen metabolism suggested to some that the process might be
restricted to this area, but this idea was soon abandoned as further reports began
to appear implicating enzymes that act in other pathways (8, 9). In particular, the
studies by Lester Reed and his associates (10, 11) showing that pyruvate dehy-
drogenase is regulated by phosphorylation-dephosphorylation served to broaden
people’s perspective regarding the scope of the process. At about this same time
evidence was obtained that the enzyme that catalyzed the phosphorylation and
activation of phosphorylase kinase was a cyclic AMP-dependent protein kinase
that could catalyze the phosphorylation of other proteins, thus making it a proba-
ble mediator of the diverse actions of cyclic AMP (12, 13). These events served
to stimulate general interest in the process of protein phosphorylation, and during
the next fifteen years numerous enzymes were found to be regulated in this
manner. In addition, many important functional proteins other than enzymes
were shown to undergo reversible phosphorylation and to be controlled by this
process. More detailed accounts of early work on protein phosphorylation have
been reviewed elsewhere (14, 15).
II. Protein Phosphorylation-Dephosphorylation Reactions
Phosphoproteins are formed in cells through the action of protein kinases, and
they also occur as transient intermediates formed as part of the mechanism of
I. ENZYMOLOGY OF CONTROL BY PHOSPHORYLATION 5
action of certain enzymes and transport proteins [reviewed in Ref. (16)]I.t is
with the former type of protein phosphorylation that the various chapters in this
volume are concerned.
A. GENERALP ROPERTIES
The reactions of protein phosphorylation-dephosphorylation involving protein
kinases and phosphoprotein phosphatases are shown in Eqs. (1) and (2).
Protein kinase
Protein + nNTP- Protein-P, + nNDP (1)
Phonphoprolein phosphalase
+ +
Protein-P, nHzO Protein nPi (2)
As is implied in Eq. (l), a given protein kinase often catalyzes the transfer of
phosphate to more than a single site in its protein substrate. In this sense,
glycogen phosphorylase, the first enzyme shown to undergo phosphorylation-
dephosphorylation and the prototype for many studies in this field, is unusual
inasmuch as it is phosphorylated at a single site.
In protein phosphorylation reactions it is common for a single protein to serve
as the substrate for more than one kinase. In some instances different kinases
catalyze the phosphorylation of identical sites in the protein substrate, but more
commonly each kinase has its own site “preference” since, as discussed in
Section II,C,2, protein kinases exhibit a moderately high degree of specificity for
particular amino acid sequences surrounding phosphorylatable amino acid resi-
dues. The phosphorylation of an identical site by two or more protein kinases
probably occurs due to a high degree of exposure of that site combined with the
fact that protein kinases do not exhibit absolute specificities. The most inten-
sively studied set of protein kinase reactions involving a single substrate are
those that occur with glycogen synthase, which can be phosphorylated in vitro by
no less than ten different protein kinases. It should be noted, however, that all of
the kinases capable of phosphorylating glycogen synthase in virro may not func-
tion in this manner physiologically (see Chapters 11 and 12 in this volume).
As with essentially all phosphotransferase reactions, protein kinase reactions
require divalent metal ions, Mg2 probably serving as the physiologically sig-
+
nificant cation in all instances, although manganous ions are nearly always
effective in vifro. The actual substrate for the reaction depicted in Eq. (1) is the
NTP-Me2+ complex. In some instances, however, a role for divalent ions in
addition to forming the metal-nucleotide complex has been noted. This is seen,
for example, with the cyclic AMP-dependent protein kinase, which binds free
metal ions at a separate site (17, 18).
The protein kinases constitute a very diverse set of enzymes, the total number
of which is only now beginning to be appreciated. These enzymes are commonly
regulated through their interaction with “second messengers” generated within
cells in response to hormones and other extracellular agents (see below). The
6 EDWIN G. KREBS
reactions catalyzed by the kinases are also regulated through the interaction of
metabolites with their substrates, that is, through “substrate level” control (see,
for example, Chapter 9, this volume and Chapter 2, Volume XVIII).
The phosphoprotein phosphatases appear to be smaller in total number than the
protein kinases, and they probably exhibit broader specificities. This in itself
implies that these enzymes may not be as actively involved in regulating phos-
phorylation-dephosphorylation cycles as are the kinases, since the broader the
specificity the greater the number of pathways that would be affected simul-
taneously by the regulation of a given enzyme. A more passive regulatory role
for the phosphatases as compared to the kinases is also suggested by the fact that
investigators have not discovered as many diverse forms of regulation for this set
of enzymes. With respect to the last point, however, it is possible that the
phosphatases have simply been more difficult to characterize than the kinases,
and additional regulatory mechanisms may be found in the future. These matters
are considered more fully in Chapter 8.
B. SPECIFICITFYO R PHOSPHORYDLO NORS
IN PROTEINK INASER EACTIONS
The protein kinase reaction (Eq. 1) is written as being dependent on a nu-
cleoside triphosphate, The physiologically significant donor in nearly all in-
stances is probably ATP (19), although several protein kinases can also use GTP
effectively in vitro. The last category includes casein kinase I1 [reviewed in Ref.
(20)],a histone H1 kinase from tumor cells (21) and pp6CPrc (22). An unusual
protein kinase from rabbit skeletal muscle, which utilizes phosphoenolpyruvate
as a phosphate donor was reported by Khandelwal et al. (23).T he kinase studied
by this group was found to be activated by CTP (24).P hosphoenolpyruvate has
long been known to serve as the phosphoryl donor in the phosphorylation of the
enzyme involved in the first step of sugar transport in bacteria [reviewed in Ref.
(25)],b ut this process belongs in a category set apart from typical protein kinase
reactions as previously indicated.
C. PROTEINS UBSTRATSEP ECIFICITOYF PROTEINK INASES
AND PHOSPHOPROTEPIHNO SPHATASES
1. Amino Acid Residues That Serve as Acceptors
of Phosphoryl Groups
Most of the acid-stable protein-bound phosphate found in cells is present as
phosphoserine and phosphothreonine and is formed as a result of the action of
protein serine and threonine kinases. A much smaller fraction, usually less than
0.2% of the total, is present as phosphotyrosine and arises as a result of the action
of protein tyrosine kinases [reviewed in Ref. (26)].T he possible existence of a
1. ENZYMOLOGY OF CONTROL BY PHOSPHORYLATION 7
hydroxylysine kinase has also been indicated (27). In addition to protein kinases
that catalyze the previously mentioned phosphorylations, there are indications
that protein kinases exist that can catalyze the formation of acid-labile phos-
phohistidine and phospholysine in proteins (28-30). No work has been done on
phosphatases that reverse the action of this last set of kinases, so it is not known
whether such phosphorylations represent reversible protein modifications.
2. Structural Determinants of Specificity
The phosphorylation site sequences in various protein substrates for a number
of different protein kinases have been determined and investigators using syn-
thetic peptides as substrates have also elucidated a number of the structural
requirements for substrate specificity of protein kinases. It has become clear that
although the primary structure of a phosphorylation site sequence does not tell
the whole story, it is usually possible to distinguish differences between the site
specificities of the various kinases. For the CAMP-dependent protein kinase, for
example, it can be predicted that if a given protein has an exposed -Arg-Arg-X-
Ser-X- sequence, it will be phosphorylated by this enzyme. Similarly, casein
kinase I1 phosphorylates exposed serine (or threonine) residues followed by one
or more glutamic acids residues one position removed from the serine (e.g.,
-X-X-Ser-X-Glu-). The entire specificity pattern of a protein kinase is not re-
vealed, however, simply by knowledge of its preferred primary amino acid
sequence. Higher orders of protein structure also play a part in determining
whether a given protein can be phosphorylated at an appreciable rate by a given
kinase. These considerations are discussed in chapters that refer to specific
kinases.
The specificity of the phosphoprotein phosphatases is not as well understood
as that of the kinases. One of the problems in studying this set of enzymes has
been that earlier investigators were never certain whether their preparations
contained more than one enzyme, and conclusions regarding specificity were
obviously difficult to reach. Later studies, however, and particularly in the
laboratory of Dr. Philip Cohen, made considerable headway in defining and
classifying these enzymes. The relative activities of different phosphatases to-
ward a number of different phosphoprotein substrates have been determined.
Several general observations can be made: First, there is probably more overlap-
ping of specificity among the phosphatases than is seen for the kinases. Second,
it is evident that the pattern of specificity is not one in which a given phosphatase
is “designed” to dephosphorylate a set of proteins phosphorylated by a given
kinase. Thus, although the kinases recognize specific amino acid sequences in
their substrates, this is not so readily apparent for the phosphatases. This sug-
gests that higher orders of protein structure may be more important in governing
substrate specificity for the phosphatases than for the kinases. If this is so, then
one might anticipate that regulation of phosphatase activity might commonly
8 EDWIN G. KREBS
occur at the “substrate level” (i.e., through the interaction of metabolites with
the phosphoproteins involved). Through such interactions and the ensuing con-
formational changes phosphatase activities could be regulated in a highly specific
manner. Finally, it is apparent that some phosphatases exist that may attack low-
molecular-weight substrates as well as proteins under physiological conditions.
Synthetic phosphopeptides have been employed only rarely in studies on phos-
phoprotein phosphatases. A pioneering effort in this regard was the work of
Titanji et al. (31).
D. THER EVERSIBILITOYF PROTEINK INASER EACTIONS
The phosphorylation reactions that result in the formation of phosphoserine
and phosphotyrosine in proteins, and presumably those that result in phos-
phothreonine, can all be demonstrated to be reversible albeit not under physio-
logical conditions. Rabinowitz and Lipmann (32) were the first to describe the
reversibility of protein phosphorylation. Using phosphorylated phosvitin as a
phosphate donor, they demonstrated the formation of ATP from ADP in the
presence of phosvitin kinase (probably casein kinase 11) from yeast or brain and
postulated that runs of adjacent phosphoserines in phosvitin may have accounted
for the apparent high free energy of hydrolysis of the bound phosphate in this
protein. That such structural relationships are not essential for reversibility of
protein kinase reactions was brought out by the work of Shizuta et al. (33),u sing
the cyclic AMP-dependent protein kinase as enzyme and substrates in which no
adjacent phosphoserines are present. These workers determined the equilibrium
position in a well-defined protein kinase reaction and calculated that the free
energy of hydrolysis of serine phosphate in this substrate (32P-labeled reduced
carboxymethylated maleylated lysozyme) was -6.5 kcal mol- l. The rever-
sibility of a number of other protein serine kinase reactions has been reported
(34-37). Fukami and Lipmann (38) described reversibility of Rous sarcoma-
specific immunoglobulin phosphorylation catalyzed by the src gene kinase and
reported a free energy of hydrolysis of -9.48 kcal mol-I for protein-bound
tyrosine phosphate, appreciably higher than the value of -6.5 kcal reported for
protein serine phosphate by Shizuta et al. (33). However, the latter workers
assumed a different AGO’ for hydrolysis of ATP (-8.4 kcal mol-l) than that
used by Fukami and Lipmann (- 10 kcal mol- I). When the same value is used,
a free energy of hydrolysis of -8.1 kcal mo1-I is obtained for protein serine
phosphate (i.e., only slightly lower than that for tyrosine phosphate). A standard
free energy of hydrolysis for serine phosphate in pyruvate kinase was found to be
-6.6 kcal mol-I by El-Maghrabi et al. (37). These workers used -8.4 kcal
mol-I for the free energy of hydrolysis of ATP.
Very little has been made of the possible physiological significance of the
reversal of protein kinase reactions, but it is possible that under some circum-
stances the process could be important. The fact that protein-bound serine phos-
