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