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CHAPTER-I
INTRODUCTION
INTRODUCTION
Phosphorylation - dephosphorylation of proteins is one of
the most widespread and important covalent modifications involved
in cellular regulation. The presence of phosphate, tightly
associated with protein was known since the late nineteenth
century, and hints that this phosphate might be covalently linked
were obtained in 1906. However, the first phosphoaminoacid was
isolated only in 1933 (Hunter and Cooper, 1985). The regulatory
mechanism of protein phosphorylation was identified in the mid
1950's by Fischer, Krebs and Sutherland during their studies on
the control of glycogen metabolism (Krebs and Fischer, 1956;
Wosilait and Sutherland, 1956; Krebs et al., 1959; Freidman and
Larner, 1963) . The two forms of glycogen phosphorylase-· a' and
'b' (Cori and Green, 1943; Cori and Cori, 1945) were shown to be
the phosphorylated and nonphosphorylated forms of the same
protein. The kinase and phosphatase which are involved in the
interconversion of these two forms are in turn regulated by phos
phorylation - dephosphorylation (Fischer and Krebs, 1955;
Sutherland and Wosilait, 1955; Krebs et al., 1959). Another
enzyme, glycogen synthase was also shown to be controlled in a
similar manner (Freidman and Larner, 1963). Since then it has
been revealed that there are many key regulatory proteins in
normal cells which exist in either phosphorylated or
dephosphorylated forms. The steady state level of phosphorylation
of these proteins depends on the relative activities of the
1
protein kinases and protein phosphatases. By the 1980s it became
evident that protein phosphorylation is one of the essential
mechanisms by which many cellular functions such as cell
division, growth, differentiation, membrane transport, secretion,
signal transduction, neurotransmission and even memory are
regulated (Cohen 1988}.
1. 1. General properties of the protein phosphorylation
dephosphorylation reaction:
The phosphorylation - dephosphorylation reactions are shown
in equations (1} and (2}:
Protein kinase Protein + nNTP ~================= Protein-Pn + nNDP
Protein-Pn + nH2o
Phosphoprotein phosphatase
Protein + nPi
(1}
(2)
The phosphorylation reaction involves transfer of phosphate
group from a phosphoryl donor to an amino acid residue of the
protein and the reaction is catalyzed by protein kinases. ATP is
the preferred phosphoryl donor in the physiological conditions.
However, in vitro studies have shown that several protein kinases
can utilize GTP as effectively as ATP. A protein kinase from the
rabbit skeletal muscle was found to be activated by CTP (Mateo,
1984) .
Protein kinase reactions, like all phosphotransferase
reactions, require divalent metal ions. Mg2+ is probably the
2
cation of choice under physiological conditions, although Mn2+
ions are also almost as effective as Mg2+ in in vitro conditions.
The actual substrate for the kinase reaction is the ·nucleoside
triphosphate-metal ion' complex.
The amino acids to which the phosphate is mostly transferred
are serine and threonine. In a typical eukaryotic cell about 90%
of phosphorylation is at serine, and about 10% at threonine.
Phosphorylation at tyrosine is a very rare event and in general
phosphotyrosine constitutes about 0.05-0.2% of the total protein
phosphorylation (Hunter, 1984; Hunter and Cooper, 1985). Inspite
of being minor components of the cell, phosphotyrosine containing
proteins and tyrosine kinases have been speculated to have
crucial role in many cellular processes. Apart from serine,
threonine and tyrosine other residues like histidine and lysine
are also known to have covalently bound phosphates (Smith et al.,
1974; 1978).
The dephosphorylation reactions are catalysed by phospho
protein phosphatases. Both phosphorylation and dephosphorylation
reactions are known to be reversible, but the physiological role
of this reversibility is not clear. For performing a regulatory
function the addition or removal of a phosphate moiety must be
precisely controlled, i.e. the activity of protein kinases and
phosphatases must be regulated. Autophosphorylation is one of the
major mechanisms by which most of the protein kinases are regula-
3
ted. Though the actual role of autophosphorylation is not yet
fully understood, there is evidence that autophosphorylation
causes marked enhancement of the protein kinase activity in many
of the kinases examined so far (Rosen et al., 1983; Weinmaster et
al., 1984; Swarup and Subrahmanyam, 1985; Ellis et al., 1987;
Herrera and Rosen, 1986; Weinmaster and Pawson, 1986; Swarup et
al., 1988). The prevailing concept with respect to autophospho
rylation reaction of protein kinases is that many of these
enzymes may be maintained in an inactive state as a result of the
interaction between their autophosphorylation sites and their
protein substrate binding sites(Krebs, 1986). It is possible that
autophosphorylation may result in a conformational change that
allows the catalytic site a better access to exogenous
substrates. (Hanks et al., 1988).
1.2. Classification of protein kinases:
The classification of enzymes is generally based on the kind
of chemical reactions they catalyze. The individual enzymes are
then delineated and named on the basis of the specific substrate
on which they act. The protein kinases cannot be classified by
this system because a given protein kinase usually catalyzes the
phosphorylation of a number of different proteins. Moreover, in
some cases a single protein can serve as a substrate for more
than one kinase. It is also known that the same site can be
phosphorylated by two or more different kinases.
4
The protein kinases can be broadly divided based on whether
the amino acid acceptor of the phosphoryl residue is:
(1) Protein alcohol group - the protein serine and protein
threonine kinases.
(2) Protein phenolic group - the protein tyrosine kinases.
(3) Protein nitrogen containing groups - the protein histidine
and lysine kinase (Smith et al., 1974).
Each main class of kinases would include individual groups of
enzymes or subfamilies on the basis of the regulation of their
activities.
1.3. Serine/threonine specific protein kinases:
An enormous amount of information is available on the
kinases which phosphorylate serine/threonine residues. Many of
the serine/threonine kinases have been purified and the genes for
several of them have been identified. Phosphorylase kinase was
the first serine/threonine kinase to be purified (Krebs et al.,
1959) and it was followed by the purification of the cAMP
dependent protein kinase in 1969. To date nearly 100
serine/threonine kinase have been identified, mainly by the use
of molecular cloning techniques.
The role of some of the serine/threonine kinases such as the
kinases involved in glycogen metabolism and cAMP dependent
protein kinase are well known. There is evidence indicating that
the products of two of the genes involved in the cell cycle are
5
serine/threonine kinases (Reed et al., 1985; Simanis and Nurse,
1986). The action of one of these, the cdc2+ protein is known to
be regulated by a serine/threonine kinase wee1+, which in turn is
regulated by yet another serine/threonine kinase (Russel and
Nurse, 1987). The combined action of these genes indicate that
the process of cell division might be regulated by a cascade
involving a series of protein kinases.
The members of the protein serine/threonine kinase family
have been divided into 9 subfamilies: Table I (Hanks et al.,
1988) . Another system of classification by Hunter, ( 1987) has
grouped these kinases according to the source of their origin.
About 50 protein serine/threonine kinases have been listed from
mammalian systems, 9 from Drosophila and 14 from yeast.
1.4. Tyrosine-specific protein kinases:
The first indication of the presence of a kinase that could
phosphorylate tyrosine residues arose from the observations of
Eckhart et al., in 197Q,. Phosphorylation at tyrosine residues
could not be detected earlier because phosphotyrosine was usually
masked by phosphothreonine when conventional methods of separa
tion were used. After the development of suitable separation
techniques for phosphotyrosine, it became evident that low levels
of phosphotyrosine were present in all animal cells (Hunter and
Sefton, 1980; Sefton et al., 1980). Since then, due to the
intensive search, the number of tyrosine kinases discovered has
6
A.
B.
c.
D.
E.
F.
G.
TABLE-I
Protein-serine/threonine kinases
Subfamily Serine/threonine kinase
Cyclic nucleotide-dependent subfamily
Calcium-phospholipid-dependent subfamily
Calcium-calmodulin-dependent subfamily
SNFl subfamily
Casein kinase subfamily
CDC28-cdc2+ subfamily
Raf-Mos proto-oncogene subfamily
7
1. c-APK-a 2. c-APK- B 3. SRA 3 4. TPKl (PK25) 5. TPK2 6. TPK3 7. cGMP dependent protein
1. PKC- a 2. PKC- B 3. PKC-Y 4. PKC-e: 5. DPKC
1. CaMII- a 2. CaMII- B 3. PhK- y 4. MLCK-K 5. MLCK-M 6. PSK-Hl 7. PSK-C3
1. SNFl 2. ninl+ 3. KINl 4. KIN2
1. CKII- a 2. DCKII
1. CDC28 2. cdc2+ 3. CDC2Hs 4. PSK-J3 5. KIN28
1. Raf 2. A-Raf 3. PKS 4. Mos contd •.•
kinase
Table-r continued ...
H.
I.
Subfamily
STE 7 subfamily
Members with no close relatives
Reference Hanks et. al., (1988)
7a
Serine/threonine kinase
1. STE 7 2. PBS 2
1. CDC 7 2. wee 1+ 3. ran 1+ 4. PIM-1 5. HSVK
increased at an accelerated pace. Many of these enzymes have been
speculated to be involved in cellular activities like cell growth
and differentiation.
All the known tyrosine kinases were classified into three
groups (Swarup et al., 1984, Hunter and Cooper, 1985). (1) Those
associated with retroviral transforming gene products and their
cellular homologues. ( 2) The tyrosine kinases known to be
stimulated by growth factors. (3) The tyrosine kinases which do
not belong to the above two classes. There is no clear distinc
tion between the retroviral and receptor tyrosine kinase genes.
Some of the retroviral oncogenes are known to have been derived
from endogenous genes encoding growth factor receptors. For
example, it is known that the oncogene v-erb B is derived from
the epidermal growth factor receptor gene (Downward et al., 1984;
Ullrich et al., 1984) . The v-fms might also have been derived
similarly from the receptor of colony stimulating factor-1 (Sherr
et al., 1985), and the oncogene neu* is known to be related to
v-erb B (Bargmann et al., 1986a, 1986b).
Hanks et al., (1988) have subdivided the protein-tyrosine
kinases into six subfamilies.
A) Src subfamily
B) Abl subfamily
C) Epidermal growth factor receptor subfamily
D) Insulin receptor subfamily
8
E) Platelet-derived growth factor receptor subfamily
F) Other receptor-like protein-tyrosine kinases
Table-!! lists the protein tyrosine kinases falling in each of
the subfamilies. With the discovery of new protein-tyrosine
kinases, the number of subfamilies is likely to increase in
future.
Among the lower organisms, low levels of protein tyrosine
kinase activity has been detected in yeast extracts (Schieven et
al., 1986) . But despite strenuous efforts no enzyme with this
activity nor a gene for the tyrosine kinase could be isolated. In
the photosynthetic bacterium Rhodospirillum rubrum also there is
evidence of tyrosine kinase activity (Vallejos et al., 1985). The
protein kinase genes from Drosophila melanogaster includes
several protein-tyrosine kinase genes as well as protein-serine
kinase genes.
Based on these observations it has been stated that the
protein-tyrosine kinases
kinases (Hunter, 1987).
evolved later than the protein-serine
Out of a total of 79 known protein
kinases from mammalian systems, 29 are protein tyrosine kinases,
and from a total of 18 protein kinases identified from
Drosophila, 9 are tyrosine kinases (Hunter, 1987).
Conserved sequences and catalytic domain of tyrosine kinases:
There is an overall similarity among the catalytic domains
9
TABLE-II
Subfamilies of the protein - tyrosine kinases
Src Abl EGF
receptor Insulin PDGF
Others receptor receptor
92 132 135 16 .. 202 Fgr Abl EGFR
lt9 201 221t 72 INSR PDGFR FER
FYN 99 160 ARG 112 222 V-erb-B
203 39 116 191t IGF-lR CSF-lR Piml
HCK 1H 228 Dash 78 Neu 6 ItO 221 (c-fms)
Kit 225 17't DILR 131 rel ......
LCK 10,. 119 199 2o9 Nabl 63 115 0 Der
11 123 133 11t1 191 97 181 Ros Ret mil
LYN223 Fes/Fps 80 1 .. 5 11t6 68 ItS 210 1't3
Sevenless sis raf
Src 1 120 173 192 eph 77 193
TRK 121 1Sit 207 flg mos
Yes 179 elk 113 Met 19 131t Cekl 136 218
FD17
tkl176 flk 113 ltk 7 bek 107 218 FD22
Dsrc64 78 166 NCP94 Sit sea 75
Dsrc28 65 TRKll 56
TRK16 56
of protein kinases. These similarities imply that the protein
serinejthreonine kinases and the. protein tyrosine kinases have
diverged from a single ancestral catalytic domain (Hunter, 1987).
The catalytic domain is not conserved completely, but consists of
highly conserved stretches interspersed with regions of low
conservation. There are 11 such conserved subdomains (Hanks et
al. , 1988) .
The precise requirements for a competent protein-tyrosine
kinase catalytic domain have not been defined, and different
enzymes could have slight variations. The leucine on the c
terminal region corresponding to Leu-516 in pp60v-src appears to
be very important. All protein tyrosine kinases have a
hydrophobic residue at this site; either leucine or
phenylalanine (Hunter and Cooper, 1986). Substitution of the c
terminus of pp60v-src, including Leu-516 and a two amino acid
deletions of residues 502 and 504, have been shown to abolish
both protein kinase and transforming activities (Bryant and
Parsons, 1982; Wilkerson et al., 1985).
On the N-terminal either the boundary is not as clear. There
is evidence of homology among various kinases which corresponds
to position 260 onwards in pp60v-src (Cross et al., 1985). The
lysine residue at site equivalent to position 295 of pp60v-src is
conserved in all protein kinases and it is found along with the
sequence X-Ala-X-Lys where both the X residues are nonpolar.
11
Derivatization of this lysine with the ATP affinity analog,
p-fluorosulfonyl-benzoyladenosine (FSBA) inhibits the protein
kinase activity (Zoller et al., 1981; Kamps et al., 1984; Russo
et al., 1985). Based on this reaction, it may be postulated that
this lysine is in the vicinity of either 8 or y-phosphate of the
substrate ATP. Substitutions by site-directed mutagenesis seem to
abolish both the prote.in kinase activity and trans forming
activity of pp60v-src (Hannink and Donoghue, 1985; Snyder et al.,
1985; Kamps and Sefton, 1986; Weinmaster et al., 1986).
A highly conserved sequence Gly-X-Gly-X-X-Gly is found about
20 residues N-terminal to the Lys-295 of pp60v-src. This
sequence has been conserved in all protein kinases. Such a
glycine-rich region is a common feature of nucleotide binding
sites in proteins (Wierenga and Hol, 1983). A model for the ATP
binding site of the v-src based on the three dimensional
structures from other nucleotide binding proteins (Sternberg and
Taylor, 1984) shows that the Gly-X-Gly-X-X-Gly residues form an
elbow around the nucleotide with the first glycine in contact
with the ribose moiety and the second glycine lying near the
terminal pyrophosphate. The residues 385-387 of pp60v-src (Arg
Asp-Leu) also shows homology throughout the protein kinases.
The subdomain VIII described by ·Hanks et al., ( 1988)
contains many residues which are conserved in both tyrosine and
serine/threonine kinases. The triplet Ala-Pro-Glu is a key
12
protein kinase catalytic domain indicator (Hunter and Cooper,
1986). Mutagenesis studies have shown that each residue in the
Ala-Pro-Glu consensus is required for activity of v-src (Bryant
and Parons, 1983; 1984) as it lies near the catalytic site. The
subdomains VI and VIII contain residues that are specifically
conserved in either the protein-serine/threonine or the protein
tyrosine kinases and may play a role in recognition of the
correct hydroxyamino acid. In the subdomain VI, the protein
tyrosine kinase consensus is either Asp-Leu-Arg-Ala-Ala-Asn for
the vertebrate members of the src subfamily, or Asp-Leu-Ala-Ala
Arg-Asn, for all other tyrosine kinases. There is another region
highly conserved among the protein-tyrosine kinases which 1 ies
immediately on the amino terminal side of the Ala-Pro-Glu
consensus in the subdomain VIII. The protein-tyrosine kinase
consensus through this region is Pro-Ile/Val-LysjArg-Trp-ThrjMet
Ala-Pro-Glu. These regions in subdomains VI and VIII which
indicate substrate specificity have been used to pynthesize
oligonucleotide probe for screening eDNA libraries (Hanks et al.,
1988).
Regulation of tyrosine kinases:
The regulation of tyrosine kinases has not been well
characterized. In the case of growth factor receptor tyrosine
kinases it is known that the enzyme activities are regulated by
their specific ligands, whereas for the other tyrosine kinases no
physiological regulatory molecules have been identified. Also
13
there is no evidence of dependence of tyrosine kinases on any of
the cyclic nucleotides, ca2+ or calmodulin.
Autophosphorylation is one of the main regulatory mechanism
exhibited by most of the known tyrosine kinases. It usually '"'
increases the Vmax and thereby enhances the kinase activity of
many tyrosine kinases, as is seen in the case of pp60 v-src
(Graziani et al., 1983), P140gag-fps (Hunter and Cooper, 1986)
and insulin receptor (Rosen et al., 1983; Yu and Czech, 1984;
Ellis et al., 1986) . The activation of the receptor tyrosine
kinase of the EGF-R due to autophosphorylation is not conclusive
(Bertics and Gill, 1985;· Downward et al., 1985). Autoactivation
by autophosphorylation for some normal cellular tyrosine kinases
has also been reported. A tyrosine kinase of 56 kd from normal
rat spleen is shown to be activated when incubated with ATP, and
the increase in activity might be due to the autophosphorylation
of the enzyme (Swarup and Subrahmanyam, 1985, 1988). A similar
mechanism is exhibited by a bovine spleen tyrosine kinase (Kong
et al., 1988).
The tyrosine in the autophosphorylation site has been shown
to be important for the activity of tyrosine kinase. For
instance, a significant reduction of the kinase and transforming
activities of the v-src was observed on replacing the tyrosine
at the autophosphorylation site to phenylalanine (Weinmaster et
al., 1984). A similar mutation of the human insulin receptor also
14
resulted in dramatic reduction of the insulin stimulated tyrosine
kinase activity (Ellis et al., 1986). The site of autophosphory-
lation may be present in the catalytic domain or elsewhere in the
molecule. It has been observed that several of the autophosphory-
lation sites lie in the catalytic domain. Therefore, there exists
a possibility that they may have been conserved because they
serve some unidentified purpose (Hunter and Cooper, 1986).
Autophosphorylation can be intramolecular or intermolecular.
The EGF-receptor undergoes intramolecular autophosphorylation
(Weber et al., 1984; Yarden and Schlessinger, 1986), whereas by
in vitro experiments it has been demonstrated that in the case of
p140gag-fps autophosphorylation is intermolecular (Weinmaster et
al., 1986). It is speculated that autophosphorylation is mainly
intramolecular. The tyrosine residue which is involved in the
autophosphorylation reaction competes with the exogenous
substrates and after phosphorylation, the part of the protein
containing the tyrosine is displaced from the substrate binding
site, thereby allowing other proteins to be phosphorylated
(Hunter and Cooper, 1986). Though autophosphorylation enhances
the tyrosine kinase activity of most of the known tyrosine
kinases, autophosphorylation need not always be autoactivating.
The tyrosine residues other than those involved in the auto-
phosphorylation reaction could be phosphorylated by other
tyrosine kinases and may be involved in the regulation of the
15
enzyme. In the p140gag-fps protein there is a tyrosine phosphory
lation site which must be phosphorylated by another tyrosine
kinase (Weinmaster et al., 1986). In the case of pp60c-src it is
clear that phosphorylation at Tyr-527 has a crucial role in the
regulation of the enzyme. The site of autophosphorylation in
pp60c-src is the tyrosine residue at position 416, but in in
vitro conditions, it has been shown that Tyr-527 is the residue
usually phosphorylated, while the Tyr-416 may be transiently
phosphorylated. This differential phosphorylation has a very
important regulatory role in the normal cells. The in vitro
kinase activity displayed by pp60c-src is only 2-10% as compared
to pp60v-src and it also has very low transforming ability (Iba
et al. , 1984; 1985; Shalloway et al., 1984; Coussens et al. ,
1985a; Johnson et al., 1985). Phosphatase treatment brings about
an increase in kinase activity ( Courtneidge, 19 8 5; Cooper and
King, 1986) which might be due to dephosphorylation of Tyr-527
and enhanced autophosphorylation at Tyr-416. It is also seen that
in cells transformed by polyoma virus, association of pp60c-src
with polyoma middle T antigen brings about phosphorylation of
Tyr-416 and not Tyr-527, and there is an increase of at least 10-
fold in the in vitro kinase activity (Bolen et al., 1984;
Courtneidge and Smith, 1984; Courtneidge, 1985; Cartwright et
al. , 1986) • Mutants which have enhanced kinase and transforming
activities are also phosphorylated in vivo at Tyr-416 and not at
Tyr-527 (Iba et al., 1985; Levy et al., 1986). These observations
16
and the results of the site directed mutagenesis of tyrosine at
positions 416, 519 and 527 have conclusively proved that in vivo
phosphorylation of Tyr-527 is a mechanism by which pp60c-src is
negatively regulated in normal cells. It has also been shown that
in the process of cellular transformation by pp60c-src, phospho
rylation of Tyr-416 and lack of phosphorylation of Tyr-527 is
essential (Cartwright et al., 1986; Kmiecik and Shalloway, 1987);
Piwnica-Worms et al., 1987). The cellular homologues of other
oncogenes like c-fps, c-abl, c-fgr and e-yes may also be
regulated in a similar way, since 'the carboxy termini of all
contain a tyrosine residue which may play an important role in
regulating the kinase activity of these molecules (Kitamura et
al., 1982; Mathey-Prevot, 1982; Naharro et al., 1984; Konopka and
Witte, 1985; Tronick et al., 1985).
The negative regulatory mechanism may be extended to the
growth factor receptors in normal cells. For instance, the EGF
receptor of fibroblasts does not show phosphorylation at tyrosine
until it binds to EGF, and this binding induces phosphorylation
at sites which are autophosphorylated in vitro even in the
absence of ligand (Decker, 1984).
Negative or positive regulatory effects can also be exerted
by phosphorylation of serine and threonine residues present in
tyrosine kinases. For example, the phosphorylation of Ser-17 of
the pp60 v-src by cAMP-dependent prote.Ln kinases increases its
17
tyrosine kinase activity (Roth et al., 1983). However, in the
case of EGF-receptor phosphorylation at Thr-654 by protein kinase
c decreases the EGF-dependent stimulation of the receptor
tyrosine-kinase (Cochet, et al., 1984; Freidman et al., 1984).
Apart from the above mentioned regulatory mechanisms there
must be other ways by which the tyrosine kinases are regulated.
Interaction of different proteins with the tyrosine kinases would
certainly have some regulatory control. In the cell lysates of
chicken embryo fibroblasts infected with cloned avian sarcoma
virus CT10, activation of several tyrosine kinase was observed.
The interpretation based on this observation is that the product
of CT10, p47gag-crk depletes negative regulatory factors, leading
to increased tyrosine kinase activity of several proteins (Mayer
et al., 1988). Recent observations based on mutation studies on
the major sites of phosphorylation of the platelet derived growth
factor receptor (PDGF-R). suggests that autophosphorylation of
Tyr-751 in the kinase region triggers the binding of activated
PDGF-R to specific cellular proteins. In this case
autophosphorylation is shown to regulate the interaction of the
PDGF-R with other cellular proteins which are thought to be
involved in the mediation of a growth signal (Kazlauskas and
Cooper, 1989). Based on these facts, it can be stated that the
tyrosine kinases would be subjected to regulation by multiple
mechanisms.
18
Knowledge of control of the regulation of tyrosine kinases
at the level of transcription is not well established. However, a
translational control has been shown to exist in many of the
tyrosine kinases. In most of the proto-oncogenes coding for
tyrosine kinases, there is an AUG codon 5' to . the authentic
initiation codon (Kozak, 1987) which has not been detected in the
oncogenes and mammalian genes in general. These 5' AUGs may be
regulating the expression of these gene by preventing the
inappropriate overexpression of the gene products that are
involved in regulation cell division and growth. Elimination of
these codons in the protooncogenes may be a common mechanism for
oncogene activation as seen for the lck protooncogene (Marth et
al., 1988).
Substrates for tyrosine kinases:
Understanding the regulation of various cellular functions
which are governed by tyrosine kinases necessitates the identifi
cation of their substrates. The methods generally used to detect
substrates for tyrosine kinases are (a) immunoprecipitation of
the phosphotyrosine containing protein using antiphosphotyrosine
antibodies, (b) alkali treatment of gels containing the proteins
from 32 P labelled cells (phosphotyrosine bonds are stable to
alkali and hence phosphotyrosine proteins can be detected) and
(c) phosphorylation of candidate substrates in vitro.
19
Inspite of the intense search for physiological substrates,
the progress has been slow. It might be because phosphotyrosine
is a rare modification and the detection of such low quantities
of proteins is inherently difficult. Moreover the substrates and
proteins phosphorylated in the in vitro condition might not be a
true representation of the phosphorylation inside the cell. Also
it is known that tyrosine kinases associated with retroviral
oncogenes are promiscuous even in vivo, and can phosphorylate
proteins which are not involved in cellular transformation
(Foulkes and Rosner 1985). Therefore, it is important to identify
physiologically significant substrates.
The specificities of the various viral tyrosine kinases for
substrates in the virally transformed cells seem to be similar
(Cooper and Hunter, 1981). The EGF and PDGF receptors also have
similar substrate specficities (Cooper et al., 1982).
Several subptrates have been identified for the viral
protein tyrosine kinases. The glycolytic enzymes - enolase,
phosphoglycerate mutase (PGM) and lactate dehydrogenase (LDH)
were found to contain phosphotyrosine when isolated from RSV
transformed chick cells (Cooper et al., 1983), but do not have
any phosphotyrosine when isolated from normal cells (Hunter and
Cooper, 1986). The cells transformed by virus containing v-yes,
v-fgr, v-fps/fes and v-abl oncogenes also show the three
glycolytic enzymes to contain phosphotyrosine (Cooper and Hunter,
20
1981b: Cooper et al., 1983). The three cytoskeletally associated
proteins, vinculin, p36 and p81: a 50 kd protein which is
associated with an 89 kd induced protein and some glycosylated
membrane proteins are amongs~he proteinsphosphorylated at .c
tyrosine by viral oncogene tyrosine kinases (Cooper and Hunter,
1983). Another phosphotyrosine containing protein found in v-src,
v-yes, and v-fps transformed chick cells is pp428 (Cooper and
Hunter, 1981a, 1981b). Cells treated with growth factors contain
several phosphotyrosine-containing proteins in addition to the
relevant growth factor receptors. These include a family of
apparently interrelated 40-45 kd proteins (Cooper et al., 1982).
A 35 kd protein which shows ca2+ dependent association with
C)' membranes is phosphorylated at tyrosine in response to EGF
~ ~~
F~ ~::~::r:::n:0
:::s~h:~y~a:i::to;:a::::o:::t::n ::s~:ii{~~~.~. '~ F:: (White et al., 1985: Izumi et al., 1987). ~-
Very few substrate molecules have been discovered for normal
tyrosine kinases. The proto-oncogene product of lck, p56lck has
been shown to phosphorylate the CD3/Ti complex in the T cells
(Barber et. al., 1989). A major cell-cycle protein the cdc2
protein kinase is shown to be the most abundant phosphotyrosine
containing protein in HeLa cells, and that its phosphotyrosine
content is regulated during cell cycle (Draetta et. al., 1988).
21
In most cases the tyrosine kinases themselves form the
substrate and get phosphorylated at the tyrosine residues either
by autophosphorylation or by other kinases.
Speculated functions of tyrosine kinases:
It is knOf.tn that cells transformed with the viruses
containing the v-src, v-yes, v-fgr, v-fps/fes and v-abl oncogenes
have a ten-fold increase in the levels of phosphotyrosine. Treat
ment of cells having ·high concentration of growth factor
receptors with their respective ligands also results in a similar
increase of phosphotyrosine. These findings indicate that
tyrosine phosphorylation plays a major role in processes 1 ike
cell transformation and growth.
The conservation of proto-oncogenes, the cellular homologues
of oncogenes, throughout .evolution implicate their importance in
the functioning of the normal cells. Slight alterations in these
molecules could activate them, leading to a transformed
phenotype. The fact that a major cell cycle control protein, the
cdc2 protein kinase has the maximum amount of phosphotyrosine is
evidence for the involvement of tyrosine kinases in cell
division.
The other tyrosine kinases like those present in terminally
differentiated cells e.g. lymphocytes, might have roles other
than regulation of cell division and growth. For instance recent
studies have revealed what might be the function of the lck
22
product. The p56lck is a tyrosine kinase which is shown to be
present associated with CD4 and CD8 as a CD4/CD8.p56lck complex
(Rudd et al., 1988; Veillette et al. , 1988) There is evidence
which indicates that the members of the CD3/Ti complex can be
phosphorylated at tyrosine by the CD4/CD8.p56lck complex (Barber
et al., 1989). This phosphorylation reaction could bring about T
cell activation (Mustelin and Altman, 1989).
1.5. Phosphoprotein phosphatases:
Depending on the state of phosphorylation, some of the key
regulatory protein kinases are present either in an active or
inactive form. The specific level of phosphorylation can be
maintained only through the combined action of kinases and
phosphatases. Phosphorylation, therefore, is a dynamic state
which depends on the amounts of the protein kinases and
phosphatases, and so it becomes essential to characterize the
phosphatases also in order to obtain a better understanding of
the phenomenon of regulation of protein activity by phosphoryla
tion.
Fewer phosphoprotein phosphatases have been characterized
than the protein kinases. Since reports in late 1940s of enzyme
activities which could dephosphorylate phosphoproteins (Harris,
1946; Feinstein and Volk, 1949), several proteins with
phosphatase activities have been identified.
23
The phosphoseryl and phosphothreonyl phosphatases:
The phosphoseryl and phosphothreonyl phosphatases have been
classified by Cohen and Ingebritsen (Cohen, 1978; Ingebritsen and
Cohen, 1983), into different groups on the basis of their
behaviour in the presence of heat stable polypeptide inhibitors 1
and 2, and their relative activities towards the aand a-subunits
of phosphorylase kinase (Ingebritsen et. al., 1980). The
phosphorylase phosphatase type 1 enzymes show susceptibility to
inhibitor 1 and 2, and can also dephosphorylate the a-subunit of
the phosphorylase kinase preferentially. The type 2-enzymes are
not susceptible to inhibitors. They have been further classified
as 2A, 2B and 2C. Phosphatase 2A is closely related to the type
1 enzyme by having broad substrate specificities and acts on the
same phosphate groups with different rates. The phosphatase type
2B is otherwise also known as Calcineurin. It is a high molecular
weight, heat labile enzyme which is stimulated by ca2+, whereas
the type 2C enzyme is a divalent metal ion dependent enzyme.
Either Mg2+ or Mn2+ can stimulate this phosphatase. All the above
mentioned phosphatases show broad specificity range (Ballou and
Fischer, 1986).
Protein tyrosine phosphatase& (PTPase):
Phosphatases which can specifically dephosphorylate phospho
tyrosine proteins were not discovered until early 1980s. Dephos
phorylation of phosphotyrosine was first detected in a human
epidermoid carcinoma cell line A-431 which has high level of EGF
24
receptors (Carpenter et al., 1979; Ushiro and Cohen, 1980). From
later studies, it was proved that unlike other phosphatases,
PTPases are enzymes which could dephosphorylate proteins which
have been phosphorylated at tyrosine residues with absolute
specificity (Brautigan et al., 1981; Foulkes et al., 1981; 1983;
Swarup et al., 1981; 1982a; 1982b; Chernoff and Li, 1983).
Several cell lines and tissues were found to have PTPase
activity (Foulkes, 1983; Shriner and Brauatigan, 1984; Swarup and
Subrahmanyam, 1989). In rabbit, among the tissues examined, kidney
was found to show the highest level of the PTPase activity
(Shriner and Brautigan, 1984). However, when PTPase activity
from particulate and soluble fractions of various tissues from
rat was examined, it was seen that spleen had the maximum
activity associated with the particulate fraction, and among the
soluble fractions examined, brain showed the highest PTPase
activity (Swarup and Subrahmanyam, 1989).
Some of the PTPases have been purified. A 36 kd PTPase was
purified by Swarup and Subrahmanyam (1989). Tonks and others have
purified a 35 kd PTPase from human placenta (Tonks et al., 1988b,
1988c) . The aminoacid sequence of the 35 kd soluble phospho
tyrosine phosphatase from placenta had similarities to the cyto
plasmic domain of the leukocyte cell surface protein CD45 and
later it was proved that the CD45 can dephosphorylate phospho
tyrosine (Tonks et al., 1988a). The CD45 is a membrane bound
25
protein; its structure is analogous to the growth factor receptor
protein-tyrosine kinases. Based on these similarities, it can be
suggested that the CD4 5 cytoplasmic domain might also be
regulated by extracellular ligands like the growth factor
receptor kinases. This implies that there might be a family of
11 receptor phosphotyros ine phosphatases 11 (Hunter, 198 9) . A
putative receptor PTPase, LAR, was isolated from a placental eDNA
library using a CD45 eDNA probe (Streuli et al., 1989a}. Two
PTPases have also been cloned from Drosophila using degenerate
oligonucleotide probes which correspond to the catalytic domain
sequence (Streuli et al., 1989b).
The role of regulation of phosphorylation events by the
phosphatases though important, has not been as extensively
studied as that of protein kinases. It is believed that there is
probably more overlapping of specificities among the phosphatase
than is seen for the kinases. It is not evident whether a given
phosphatase is 'designed • to dephosphorylate a set of proteins
phosphorylated by a given kinase, nor is it apparent whether all
the phosphatases recognize specific amino acid sequences. The
protein-serine phsphatases have been extensively studied (Cohen
1989). Among the phospho~erine phosphatases PP-2A and PP-2B have
weak activity towards phosphotyrosine. These findings indicate
that the regulation of phosphatase activity might also be
specific like that of the kinases. The recent discovery that
26
certain cell cycle genes and a transcription regulatory gene
encode protein serine phosphatases indicates that protein
phosphatases do not simply constitutively reverse the effect of
protein kinases but rather themselves play central roles in
cellular physiology (Cyert and Thorner, 1989; Hunter, 1989). The
regulation and functions of the PTPases are not clearly
understood. Unlike for the protein-serine phosphatases it is not
clear whether the PTPases have regulatory subunits. Regulation by
heat stable low molecular weight protein inhibitors is seen in
case of protein-serine phosphatases. Recently, two heat stable
protein inhibitors were identified for two of the PTPases of the
brain (Ingebritsen, 1989). The receptor PTPases might have same
type of regulation as is seen for growth factor receptor kinases,
and this would mean the presence of a new membrane signal
transduction mechanism in the cells (Hunter, 1989). The specifi
cities of the membrane bound receptor PTPases might be different
from non receptor PTPases (Tonks et al., 1989). Unlike the
protein kinases there ex~sts no homology between protein-serine
phosphatases and PTPases. But the presence of tyrosine
phosphatases in Drosophila is suggestive that there might be a
conservation of the PTPases in the course of evolution.
1.6. Rationale for the present investigation:
In general the function of non-receptor type cellular
tyrosine kinases is not known. In order to study the function of
these kinases it is essential to isolate either these proteins
27
(in purified form) or their genes or both. One of the possible
approaches for determining the function involves the use of
antisense oligodeoxynucleotides for blocking specifically the
synthesis of its gene product. Site-directed mutagenesis can also
pro~ide some information about the function of the genes. Such
approaches require the knowledge of the nucleotide sequence of
the coding region of the genes. A study of the expression of the
gene in various embryonic and adult tissues is helpful in
providing important clues about the function of the gene product.
Normal rat spleen was chosen as a model system to identify
and isolate tyrosine kinases and phosphotyrosine phosphatases
because among the various tissues of rat surveyed, the highest
level of both the tyrosine kinase and PTPase activities were seen
in spleen (Swarup et al., 1983, 1984; Swarup and Subrahmanyam,
1989). Two major.species of tyrosine kinases were identified from
the rat spleen and the 60 kd protein was purified (Swarup et al.,
1988). A protein tyrosine phosphatase has also been purified from
the rat spleen (Swarup and Subrahmanyam, 1989).
The work described in the following chapters deals with the
construction of eDNA libraries from normal rat spleen and brain
and also from the tumor AK-5, isolation of a eDNA clone related
to the src tyrosine kinase family from the spleen eDNA library
28
and the nucleotide sequence analysis of the clone. The
preliminary studies based on the expression of the rat tyrosine
kinase eDNA clone are also presented.
29