Tyrosine-specific protein kinases of normal tissues

TYROSINE-SPECIFIC OF NORMAL

PROTEIN TISSUES

KINASES

GHANSHYAM SWARUP*, JAI DEV DASGUPTA*~" and DAVID L. GARBERS*t~ Department of Pharmacology*, Physiology~ and the Howard Hughes Medical Institute Laboratoryf, Vanderbilt University School of Medicine, Nashville, TN 37232

INTRODUCTION

Phosphorylation of proteins at tyrosine residues has received great attention since its discovery in 1979 by Eckhart e t aL (1). Phosphorylation of tyrosine residues in proteins appears to be a rare modification since relatively small amounts of phosphotyrosine are found in acid hydrolysates of cellular proteins (2). Isolation and subsequent purification of the enzymes responsible for phosphorylation at tyrosine have shown that this reaction is catalyzed by specific protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) which normally do not phosphorylate at serine or threonine residues of proteins (3-10). All of the tyrosine-specific protein kinases described to date autophosphorylate at tyrosine residues and have activity independent of cyclic nucleotides or Ca 2+. The phosphorylation of tyrosine residues of proteins is a reversible event (like the phosphorylation ofserine or threonine); the dephosphorylation of proteins phosphorylated at tyrosine is carried out by phosphotyrosine-specific protein phosphatases (11-17). Some (or all) of the phosphotyrosine-specific protein phosphatases can be distinguished from the phosphoserine (or phosphothreonine)-specific protein phosphatases by the use of orthovanadate, which at micromolar concentrations appears to selectively inhibit phosphotyrosine-specific protein phosphatases (13-15). The protein bound tyrosine phosphate is a high energy linkage. The free energy of hydrolysis ( ~ G °) of protein bound tyrosine phosphate has been reported to be -9.48 Kcal in the presence of 5 mM Mg 2+ at pH 6.5 (assuming an approximate AG ° o f - 1 0 Kcal for hydrolysis of ATP) (18). Free tyrosine phosphate is not an energy rich linkage (18). Hydrolysis or formation of such a high energy tyrosine phosphate bond in proteins could conceivably bring about a conformational change in the protein resulting in an altered functional state of the molecule. In the case of the insulin receptor kinase it has been suggested (although alternative explanations are possible) that phosphorylation at a tyrosine residue causes activation of the protein kinase (19). Initially, two groups of tyrosine-specific protein kinases were described: those associated with the protein product of the viral genes (known as 267

268

GHANSHYAM SWARUP, et al.

oncogenes) responsible for cell transformation, and those associated with the receptors for growth factors (19-22). Since then, other tyrosine-specific protein kinases have been described; these are not currently known to be associated with growth factor receptors or with viral onc gene products (10, 23-27). For convenience (rather than logic) the presently known tyrosinespecific protein kinases still can be divided into two groups. The first group includes the tyrosine protein kinases associated with retroviral transforming gene products and their cellular homologs; the second group includes all other tyrosine protein kinases. The tyrosine protein kinases associated with the viral and cellular onc gene products are summarized in Table 1. Six transforming genes (out of 17 known onc genes) of avian and mammalian retroviruses code for polypeptide products which are associated with tyrosine-specific protein kinase activity (4, 28). The product of v-src gene, pp60V-~%a phosphoprotein o f Mr 60,000 has been shown to be a tyrosine-specific protein kinase (2, 5, 6). The viruses bearing fps, yes, ros, fes and abl sequences synthesize a protein (known as polyprotein) which is a fusion product of a residual gag gene and the transforming sequences (29-41). The src gene of RSV and the abl gene of Abelson murine leukemia virus have been cloned in Escherichia coli and the protein product of the cloned gene has been shown to be a tyrosine-specific protein kinase (42-44). Cloning and other experiments leave little if any doubt that the v-src and v-abl code for tyrosine-specific protein kinases. Such convincing evidence is not available for the tyrosine protein kinase activity associated with other oncogene products, although by analogy it is generally believed that the polypeptide products o f f p s , y e s , f e s and ros genes are also tyrosine protein kinases. Additionally, cells transformed by retroviruses carrying src, fps, fes, abl and yes genes show increased levels of phosphotyrosine in cellular phosphoproteins (2, 32, 38, 39, 41). Four different classes of retroviruses carrying src, fps, yes and abl gene induce phosphorylation at tyrosine residues in the same set of cellular proteins (45). Some of the cellular proteins phosphorylated at tyrosine in the transformed cells have been identified; these include vinculin (46) and three glycolytic enzymes, enolase (EC 4.2.1.11), phosphoglycerate mutase (EC 2.7.5.3) and lactate dehydrogenase (EC 1.1.1.27) (47). Whether phosphorylation of any of these glycolytic enzymes is responsible for increased glycolysis of transformed cells is not known (47). A tyrosine protein kinase activity associated with polyoma middle T antigen immunoprecipitates (which phosphorylates middle T at tyrosine residues) has been described in cells transformed by polyoma virus (I). However, ceils transformed by polyoma virus do not show elevated levels of phosphotyrosine in cellular proteins (2). Secondly, attempts to label the ATP binding site of the middle T antigen-associated kinase have been unsuccessful (48). Therefore, it is not known whether the middle T antigen itself is a tyrosine protein kinase or whether a cellular enzyme phosphorylates it.

ATP ATP

p68 gaS"r°s p120 e'agabl

p85 gag'fes p 115 gag-fes

Plasma membrane

Plasma membrane

Y73 avian sarcoma virus Esh sarcoma virus (ESV)

UR-2 virus

Abelson murine leukemia virus

Feline sarcoma virus (Snyder-Theilen strain) Gardner-Arnstein strain

v-yes

v -abl

v-fes

c-fps

c-src

V -rOs

Plasma membrane

ATP, GTP

p90 gag-yes p80 ~ag'yes

Plasma membrane

Fujinami sarcoma virus PRC II sarcoma virus PRC IV sarcoma virus Rochester sarcoma virus I (URI)

pp98 efps

pp60 c-src

p140 sag'fpS p105 gag-rp~ p170 gag'fps p1508~s-fpS

ATP

ATP

ATP

ATP, GTP

ATP ATP ATP ATP

ATP, G T P

v-fps

pp60 v'~'¢

Plasma membrane

Rous sarcoma virus (various strains)

Phosphate donor

v-src

Protein product

Subcellular location

Oncogene

Virus strain

Mn2+~>Mg 2+

Mg 2+

Mg 2+

Mg 2+

Mn 2+, Co2+>Mg 2+

Mn2+>Mg 2÷

Mn2+>Mg 2+ Mn 2+, Mg 2+

M n 2 + ) M g 2+ M n 2 + ) M g 2+ Mn 2+ Mn2+~Mg 2+

Mg 2+, Mn 2+

Cation

+

+

+

-

+

+ +

+

+ +

+

Phosphorylation o f IgG

59

52-58

39

38

40, 41

37

34, 35, 37 35, 93

29--31 32, 89, 90 91 92

2, 5, 6, 50, 51, 53 56, 88

Ref.

TABLE 1. TYROSINE S P E C I F I C PROTEIN K I N A S E S A S S O C I A T E D W I T H T H E V I R A L A N D C E L L U L A R O N C O G E N E P R O D U C T

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Most (and perhaps all) viral oncogenes have a cellular homologue called conc (28, 49). Cellular oncogenes are cellular genes which are structurally related to viral oncogenes. The protein products of some of the c-onc genes have been identified. The product of two of these c-onc genes (c-src and c-fps)

is known to be associated with tyrosine-specific protein kinase activity. The product of cellular src gene (pp6ff ~r¢) is a membrane associated phosphoprotein of Mr 60,000, associated with a tyrosine-specific protein kinase activity similar to that of pp6ff .... (50-58). The presumed product ofcfps is a 98,000 Mr protein associated with tyrosine protein kinase activity (59). The product of c-ab! gene is believed to be a 150,000 Mr protein (p. 15ff-abe) which differs in size and composition from the product ofv-ab! gene and it is not associated with tyrosine-specific protein kinase activity (60). Whether the product of c-fes gene, p92TM,a 92,000 Mr protein has tyrosine protein kinase activity, is not yet known (38). The tyrosine protein kinases not known to be associated with onc gene products can be subdivided into two groups (Table 2). The first group is represented by those protein kinases which are currently known to be stimulated by growth factors. The receptor for epidermal growth factor is a 170,000 dalton phosphoprotein which is associated with tyrosine-specific protein kinase activity towards endogenous as well as exogenous substrates (9), and the receptor itself is phosphorylated in vitro at tyrosine residues (61). However, in vivo, the receptor is phosphorylated at both tyrosine and serine residues (62). EGF stimulates phosphorylation of its own receptor and also the phosphorylation of exogenous proteins at tyrosine residues (9, 61). The EGF-dependent tyrosine protein kinase activity co-purifies with the EGF receptor and affinity labeling of the protein kinase by an ATP analog results in the labeling of a 170,000 dalton phosphoprotein as well as a 150,000 Mr proteolytic fragment of the receptor (63, 64); these and other observations suggest that the protein kinase activity is the intrinsic property of the receptor for epidermal growth factor (61, 63, 64). The receptor for insulin is also associated with an insulin stimulable tyrosine-specific protein kinase activity (22, 65-67). Insulin stimulates the phosphorylation of the 95,000 dalton subunit of its own receptor (20, 22). In vitro, the phosphorylation occurs largely on tyrosine (22); however, as with the EGF receptor, the insulin receptor is phosphorylated in vivo at both serine and tyrosine residues (65). The phosphorylation of the insulin receptor then appears to be analogous to the phosphorylation of the E G F receptor. It has not been clearly shown, however, if the tyrosine protein kinase activity associated with the insulin receptor is intrinsic or a separate protein kinase which copurifies with the receptor (67). Both EGF and insulin-stimulable tyrosine protein kinases can phosphorylate peptides having sequences similar to the tyrosine phosphorylation site of pp6ff re, and the heavy chain of IgG present in TBR (tumor bearing rabbit) serum (68-72). Thus, the protein

Spleen Brain Lung Testes Kidney T-lymphocytes Sea urchin Band 3 (erythrocytes) Leydig cells

56-53 K 58, 56-53 K 56, 53 K 58, 56, 53 K 56, 53 K 58-56 K 45, 57, 74, 84, 126 K 93 K

55, 58 K 75 K

170 K 95 K 170 K

Major endogenous phosphorylated protein

particulate microsomol, soluble particulate particulate particulate particulate particulate particulate particulate membrane particulate

membrane membrane membrane

Subcellular location of protein kinase

Mg2+>Mn 2+ Mg2+>Mn 2+ Mg 2+ Mg 2. Mg 2+ Mg2+>Mn Mg2+>Mn Mg 2+ Mg 2+

Mg 2+, Mn 2+ Mn 2+

Mg 2÷, Mn 2+ Mn 2+, Mg 2+ Mn2÷>Mg 2+

Cation

+

+ +

Phosphorylation of TBR IgG

ATP ATP ATP ATP ATP ATP ATP ATP ATP

ATP ATP

ATP, GTP ATP ATP

Phosphate donor

26 87 98

t

10 10, 27 10 10 10

23, 24 85

9, 61, 62 20, 22, 65-67 21, 73-82

Ref.

*The LSTRA kinase is included in this Table because it seems likely that this kinase is a normal cell enzyme (see Discussion). tG. Swarup, A~ R. Lawton and D. L. Garbers, Unpublished observations.

3. 4. 5. 6. 7. 8. 9. 10. 11.

Others 1. LSTRA kinase* 2. Liver

Growth Factor Stimulated 1. EGF stimulated 2. Insulin stimulated 3. P D G F stimulated

Source of kinases

TABLE 2. TYROSINE-SPECIFIC PROTEIN KINASES OF NORMAL CELLS

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kinase component of the EGF receptor kinase, insulin stimulable kinase and pp60~r~ kinase may be structurally related. Platelet derived growth factor (PDGF), the major polypeptide mitogen in serum for cells of mesenchymal origin, also has been shown to stimulate tyrosine-specific protein phosphorylation of membrane proteins of 170,000-180,000 daltons (21, 73-7.5). P D G F is a protein of 30,000 molecular weight which like insulin is composed of two nonidentical subunits linked by disulfide bonds (76-80). The pattern of phosphorylation induced by P D G F and EGF in 3T3 cells suggests that these growth factors activate different protein kinases (81). In 3T3 cell membranes it appears that EGF and P D G F stimulate the phosphorylation of a synthetic peptide through distinct receptors (75). Several lines of evidence suggest that ~ 170,000 dalton protein phosphorylated in response to P D G F is the P D G F receptor (75). By analogy with EGF it can again be suggested that the P D G F receptor is a tyrosine protein kinase (82). In addition to these growth factor stimulable tyrosine protein kinases, various other tyrosine protein kinases have been recently described which are not known to be stimulated by growth factors. A protein kinase from a lymphoma cell line, LSTRA (induced by Moloney murine leukemia virus), phosphorylates an endogenous protein of 55-58,000 daltons (pp55 or pp58) at tyrosine residues (23, 24). The protein kinase can also phosphorylate s r c related peptides (23). Two dimensional tryptic peptide mapping of phosphopeptides has shown that the phosphorylation site in pp58 is very similar to or identical with the phosphorylation site ofpp60 V-Src(83). However, the partial proteolysis maps of the two proteins are different (83). Various antisera containing antibodies to pp60v.... do not precipitate or inhibit the tyrosine protein kinase activity of LSTRA ceils (24). The protein kinase has been purified 100-fold and the partially purified preparation retained pp58 (84). By analogy to other tyrosine protein kinases, which autophosphorylate at tyrosine, it seems likely that pp58, itself, is a tyrosine protein kinase. Another lymphoma cell line MBL2 (also induced by Moloney murine leukemia virus) does not contain significant levels of pp55 or other alkalistable phosphoproteins (24). A tyrosine protein kinase activity of Mr 75,000 has been described in the soluble and microsomal fraction of rat liver using [ValS]angiotensin as a substrate for measuring tyrosine protein kinase activity (85). The partially purified preparation showed the presence of a phosphoprotein of Mr 75,000 and again this could be the protein kinase itself. Although tyrosine phosphorylation has been implicated in the regulation of cell growth and differentiation, there are very few studies on tyrosine phosphorylation in developing embryos. The presence of phosphotyrosine has been reported in plasma membranes of sea urchin eggs (86). Dasgupta and Garbers (26) have shown that tyrosine-specific protein kinase activity

273

T Y R O S I N E - S P E C I F I C P R O T E I N KINASES

increases in the sea urchin embryo within one hour after fertilization and continues to increase until at least the early gastrula stage. In addition, some stage-specific phosphoproteins appeared to be phosphorylated at tyrosine. A tyrosine-specific protein kinase phosphorylates Band 3, the anion transport protein of the erythrocyte membrane (87). The phosphorylation site in Band 3 contains mostly acidic residues (Glu, Asp) like the phosphate acceptor sites in the transforming proteins (94-97) of some retroviruses. Purified Leydig cells from rat testes have been shown to have fairly high levels of tyrosine protein kinase activity as measured by the phosphorylation of a synthetic peptide (98). In a recent report from our laboratory, Swarup et al. (10) have carried out a systematic study on the activity of tyrosine-specific protein kinases in various rat tissues using a synthetic peptide, Glu-Asp-Ala-Glu-Tyr-Ala-Ala-ArgArg-Arg-Gly (E~IGI), as a substrate. Spleen showed the highest activity followed by lung. Other tissues had 10-20 times lower activity than spleen (Fig. I). The particulate enzyme from spleen was solubilized and partially purified to a state where only a phosphoprotein doublet of Mr 53,000 and 56,000 was seen on SDS gels. Interestingly, an alkali-stable doublet

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FIG. 1. Distribution of tyrosine specific protein kinase activity in the particulate fraction of various tissues and cells. Tyrosine protein kinase activity was estimated by the phosphorylation of E~ tGa (1 mM) as described under Materials and Methods. The nonionic detergent NP-40 (0.05%) was added in all the assays except in the case of sea urchin embryos where 0.05% Lubrol W X was used.

274

GHANSHYAM SWARUP, et aL

(containing mostly phosphotyrosine) was present in the particulate fraction of several tissues including lung, brain, kidney and testes. Here, we further characterize the tyrosine protein kinase activity present in the particulate fraction from rat spleen. In particular, we examine the possible relationship of this protein kinase to the protein kinase activity of pp60~r<

MATERIALS

AND METHODS

Materials. TBR serum was obtained from Transformation Research, Inc. Orthovanadate was from Fisher. The peptide E11GI was synthesized by Peninsula Laboratories, Inc., San Carlos, CA. [7-32p]ATP was synthesized by the procedure of Walseth and Johnson (99). Bovine gamma globulin was from BioRad. D E A E Sepharose and Sephacryl S-200 were obtained from Pharmacia Fine Chemicals. All other reagents were of highest grade and were purchased from Fisher or Sigma. Tyrosine protein kinase assay. Tyrosine-specific protein kinase activity was estimated by the use of a synthetic peptide, E I 1G ~(see Table 3 for its structure) which has a sequence similar to but not identical with the major phosphorylation site in pp6(r rc. A typical assay was carried out in a vol of 50/AI containing 50 mM Tris-Cl, pH 7.8, 10/AM orthovanadate, 50 mM MgC12, 1 mM El ~G1 and 2-20 #g of protein. Wherever indicated the nonionic detergent NP40 was included in the assay at a concentration of 0.05% or 0.1%. After incubation for 10 min at 30°C the reaction was stopped by the addition of 150 /AI of 3.3% trichloroacetic acid and the assay was continued as described elsewhere (10, 27). Orthovanadate (10/AM) was included in all of the assays to inhibit the degradation of ATP and the dephosphorylation of phosphopeptide. The assay was linear with time for at least 15 rain and with protein concentrations up to at least 20/Ag/tube (particulate fraction) or 40 #g/tube (soluble fraction). Phosphorylation of IgG. Phosphorylation of antibodies (anti pp6@ rc or bovine gamma globulin) by the partially purified tyrosine protein kinase from spleen was carried out in a total vol of 50 #1 containing 25 mM Hepes, pH 7.0, 10 mM MgC12, 10/AM orthovanadate, 10-20/Ag o f l g G and 2 ~zg of the protein kinase preparation. After incubation for 30 min at 0°C [7-32p]ATP (4,000 cpm/pmol) was added to a final concentration of 20 taM and incubated for 5 min at 30°C. The reaction was stopped by the addition of 50 #1 of a solution containing 6% Na dodecyl SO4, 40 mM Tris-HCl, pH 7.5, 200 mM dithiothreitol and 25% glycerol. These samples were then heated for 5 min at IO0°C. Polyacrylamide gel electrophoresis and autoradiography were carried out as described by Swarup and Garbers (100).

TYROSINE-SPECIFIC PROTEIN KINASES

275

Purification of IgG on protein A-Sepharose. The lyophilized antiserum containing antibodies to pp60 ~rc was dissolved in 1 ml of 0.1 M phosphate buffer, pH 8.0, and applied to a column (1.0 × 5 cm) of protein A Sepharose equilibrated with 0.1 M phosphate buffer, pH 8.0. The column was washed with the same buffer until no protein was detectable in the effluent. Total IgG were then eluted with 0.1 M acetic acid containing 0.15 M NaC1; 3.0 ml fractions were collected. The peak fractions containing IgG were neutralized and then dialyzed against 5 mM Hepes, pH 7.0, and lyophilized and stored at -35°C. Purification of tyrosine protein kinase from the particulate fraction of rat spleen. Purification of tyrosine protein kinase from the particulate fraction of rat spleen was monitored by the use of a synthetic peptide, E l,Gl, as substrate. The purification was essentially as described by Swarup et al. (10). Briefly, the solubilized protein kinase (100 ml) was applied on a 1.5 × 15 cm column of D E A E Sepharose. The protein kinase activity was then eluted with 100 mM NaCI. The peak enzyme fraction from this column was further purified on a Sephacryl S-200 column (1.5 × 87 cm). The protein kinase-active fractions were dialyzed against 25 mM Hepes, pH 7.0 containing 50% glycerol, 1 m s dithiothreitol and 1 mM EDTA, and were then stored at -15°C.

RESULTS

We have synthesized peptides having amino acid sequences similar to the tyrosine phosphorylation site ofpp60 ~rcand have shown that they can be used as substrates for the assay oftyrosine-specific protein kinase activity of cells or tissue homogenates. We do not know if the pepticle successfully measures all of the tyrosine protein kinase activity present in a particular cell or tissue. Using the peptide, EI LGI, as substrate we measured tyrosine protein kinase activity of various rat tissues and found that spleen had the highest activity of the tissues we studied. We have partially characterized the soluble and particulate enzymes from spleen. The protein kinase from the soluble fraction was not stimulated by cyclic AMP, cyclic G M P or Ca z+. E G T A had no effect on protein kinase activity. In this respect, the tyrosine protein kinase activity was similar to other tyrosine protein kinases described in the literature. Most of the detergents tested had no stimulatory effect on the protein kinase activity; the exception was Lubrol WX. The tyrosine protein kinase activity from the particulate fraction of spleen could be solubilizecl by NP-40 (5%) at high pH (pH 9.0). At pH 7.0 or 8.0, 35% and 60% of the enzyme was solubilized, respectively, compared to 90-100% solubilization at pH 9.0. The solubilized enzyme was purified on D E A E Sepharose followed by Sephacryl S-200 gel filtration, and this partially AER 22--$

276

GHANSHYAM SWARUP, et aL

purified enzyme was used to study some of its properties. The tyrosine protein kinase preparation could phosphorylate the IgG (heavy chain) present in the serum from a tumor bearing rabbit; this antiserum is known to contain antibodies directed against pp60 ~rc (and some viral proteins) (Fig. 2A). Control IgG (bovine gamma globulin) could not be phosphorylated by the spleen protein kinase preparation. Both M n~+ (1 raM) and Mg 2+ (10 mM) could serve as divalent cations for the phosphorylation of the IgG by the protein kinase from spleen, although Mg2+ was more effective. However, the spleen tyrosine protein kinase activity could not be precipitated by the TBR antiserum. In this respect, the tyrosine protein kinase activity is similar to the EGF and insulin receptor associated protein kinases which can phosphorylate IgG but are not precipitated by the antiserum (70-72). It has been previously shown that the tyrosine protein kinase from spleen can phosphorytate synthetic peptides which are related to the tyrosine phosphosylation site in pp60"-s~ (10). We have tested some other peptides as substrates which do not appear to be related to the phosphorylation site in pp60v-~rc. As compared to EliG1, all other peptides (Table 3) showed lower activity with partially purified or crude, particulate spleen enzyme. [D-Trp 11] neurotensin, (TyrS)-substance P and a-neo-endorphin 1-8 showed higher relative activity with the particulate enzyme than with the partially purified enzyme. It seems likely, therefore, that the enzyme present in the membrane has a somewhat different substrate specificity than the solubilized, partially purified enzyme.

DISCUSSION

Over the past few years the products of 6 transforming genes (there are about 17 such genes known) of various RNA tumor viruses have been identified which are associated with tyrosine-specific protein kinases (see Table 1). Cells transformed by the viruses carrying these genes show increased levels of phosphotyrosine as compared to uninfected cells, and it has been suggested that the protein kinase activity ofpp60 ~rc(and perhaps of some other analogous products) is responsible for cell transformation (2-6). However, little is known about the mechanisms by which this tyrosine protein kinase activity of pp60~r~ induces cell transformation. The function of tyrosine protein kinases in normal cells is also not understood. We have estimated the levels of tyrosine protein kinase activity in the particulate and soluble fractions of various rat tissues and have found that spleen has very high activities of the enzyme. T-lymphocytes from human peripheral blood also have high levels of the enzyme, and mixed populations of lymphocytes isolated from rat spleen showed nearly as high tyrosine protein kinase activity as that of spleen. It was somewhat surprising that thymus, which has proliferating lymphocytes, showed very low levels of activity. About 85% of

abcde FIG. 2A. An autoradiograph showing the phosphorylation of the heavy chain of IgG present in TBR serum by partially purified tyrosine protein kinase from particulate fraction of rat spleen. Phosphorylation of protein A Sepharose column purified antibodies (20 pg) was carried out as described under Materials and Methods. Phosphorylated samples were subjected to polyacrylamide gel electrophoresis in presence of 0.1% Na dodecyl-SO, followed by autoradiography. (a) an&c antibody +, kinase -; (b) no antibody, kinase +; (c)an&c antibody +, kinase +; (d) control antibody +, kinase -; (e) control antibody +, kinase +.

..~_32pi

~-P-Ser ~- P-Thr

~ -

®

P-Ty r

origin

FIG. 2B. An autoradiograph showing phosphoamino acid analysis of 32 P-labeled TBR IgG heavy chain. The IgG was removed from the gel and extracted, hydrolyzed and analyzed by electrophoresis at pH 3.5 as described previously (10).

Arg-Tyr-Leu-Pro-Thr

G lu-Asp-Ala-G lu-Tyr-A la-Ala-Arg-Arg-Arg-G ly Tyr-Arg Arg-Arg-Pro-Tyr-Ile-Leu pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-D-Trp-Ile-Leu Arg-Pro-Lys-Pro-G ln-Gln-Phe-Tyr-G ly-Leu-Met-NH2 Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Arg

Structure

2

100 1 5 67 33 18

1

100 1 6 26 6 7

(%)

(%)

Protein kinase activity with each peptide was determined at 1.0 mM concentration using 60 #M ATP, 50 mM Mg 2+ and 0.05% NP-40.

EIIG! Kyotorphin Neurotensin 8-13 (D-Trpll)Neurotensin (Ty___rs)-Substance P a-Neo-Endorphin 1-8 (Porcine) Proctolin

Peptide

purified

particulate

Tyrosine protein kinase activity

TABLE 3. PHOSPHORYLATION OF src UNRELATED PEPTIDES BY THE PARTICULATE TYROSINE PROTEIN KINASE FROM SPLEEN

t,~

6~

7~ t'~

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GHANSHYAM SWARUP, et

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the lymphocyte population in thymus consists of immature T lymphocytes of various stages of development, however, and thus these observations may suggest that tyrosine protein kinase activity increases during maturation o f T lymphocytes. The inability of TBR serum to precipitate the tyrosine protein kinase isolated from the particulate fraction of spleen suggests that the enzyme is immunologically not related to pp60 VSrc,although the phosphorylation of IgG from TBR serum by this kinase suggests that the catalytic domain of the enzyme may have similarities to the catalytic site of pp60 v-src. These observations raise the question of whether the tyrosine protein kinase from the particulate fraction of spleen is the product of the cellular homolog of src gene (pp6ff'sr¢). This spleen protein kinase and pp60 ~rc kinase share some c o m m o n properties: (a) both phosphorylate tyrosine residues of casein and of IgG heavy chains of TBR serum; (b) both show a similar subcellular distribution of activity; (c) both have similar molecular weights; (d) both use Mg z+ and Mn 2+ as divalent cations; and (e) both can be phosphorylated under appropriate conditions at tyrosine and serine residues; partially purified preparations autophosphorylate at tyrosine. These observations suggest a relationship between these two proteins, and since some TBR sera are known to react with pp60 V.... but not with pp6ff .... (54) the failure of the commercial TBR serum to react with spleen protein kinase does not rule out the possibility that this protein kinase is the same as pp60C-srL Other evidence (e.g., amino acid sequence) will be required to show whether or not the spleen protein kinase is the product of c - s r c (pp6ff-src). The tyrosine protein kinase from the LSTRA cell line which phosphorylates an endogenous protein of 55-58,000 daltons may be related to the particulate tyrosine protein kinase from spleen. These two protein kinases phosphorylate endogenous proteins of similar molecular weight and the substrates can appear as a doublet (10, 24); both Mg 2+ and Mn 2+ can serve as metal ions although Mg z+ gives a higher Vmax with synthetic peptides as the substrate. A similar phosphorylated protein (pp58) has been reported to be present in normal mouse lymphocytes (83); we have found a phosphorylated doublet of Mr 56,000--58,000 in the particulate fraction of T lymphocytes from human peripheral blood (G. Swarup, A. R. Lawton and D. L. Garbers, unpublished observations). The doublet of Mr 53,000-56,000 from spleen (10) or normal T lymphocytes contains phosphotyrosine, phosphoserine and phosphothreonine; whereas a similar protein from LSTRA contains mostly phosphotyrosine. Gacon e t al. (24) have reported that the Mr 55,000 protein containing phosphotyrosine present in the detergent insoluble matrix of LSTRA cells is not detected in the detergent insoluble matrix of normal mouse lymphocytes or lymphoma cell line MBL2 (induced by Moloney murine leukemia virus). Since the genome ofMoloney murine leukemia virus does not contain an oncogene, it has been suggested that the high level of tyrosine

TYROSINE-SPECIFIC PROTEIN KINASES

279

protein kinase activity in LSTRA must arise from the unusual expression of a cellular gene (24). Tyrosine protein kinases from very diverse cell types have been described which show certain common features, and some of these features are discussed below. Specificity of phosphorylation of tyrosine residues. Purified preparations of tyrosine protein kinases phosphorylate selectively at tyrosine residues of endogenous as well as exogenous substrates. The degree of specificity for tyrosine residues appears to be very high since even histones and casein (which have a much larger number of serine or threonine residues available for phosphorylation than tyrosine residues) are phosphorylated at tyrosine residues only. Similarly serine (or threonine)-specific protein kinases do not appear to phosphorylate at tyrosine residues even at fairly high concentrations (10, 26). This high degree of specificity provides a convenient way to classify protein kinases into two groups: (1) tyrosine-specific; and (2) serine or threonine-specific (there may also be specificity between serine and threonine). Primary sequence requirement. Tyrosine phosphorylation sites in various viral transforming proteins contain one or more glutamic acid residues close to the NH2-terminal side of Tyr in the tentative configuration of-Glu-X-XGlu-Tyr- (94, 95). It has been suggested, based on the studies on phosphorylation of various synthetic peptides as substrate for viral tyrosine kinase, that Glu at the fourth position on the NH2-terminal side of Tyr is important for the recognition of the site by the kinases (96). However, the tyrosine protein kinase activity of pp60 V.... can phosphorylate various angiotensin analogs which do not have a glutamic acid residue; one of the analogs does not have an Asp or Glu (101). A glutamic acid residue at the4th position from Tyr does not seem to be important for the recognition of the peptide by tyrosine protein kinase from spleen (10). Tyrosine protein kinases from rat brain and spleen and from sea urchin embryos can phosphorylate synthetic peptides which do not have a Glu or Asp on the NH2-terminal side of Tyr (27, unpublished results). It seems more likely that hydrogen bond donor or acceptor groups (e.g., Glu, Gin, Asn, Asp, etc.) in the vicinity of Tyr may be required rather than acidic groups. Clearly more work is required to define the primary sequence requirements for the phosphorylation by tyrosine protein kinases. ,4utophosphorylation. Most (and perhaps all) tyrosine protein kinases autophosphorylate at tyrosine residues. It is not known whether the phosphorylated enzyme represents an intermediate of the phosphotransferase reaction. If a phosphoenzyme intermediate is formed the kinetic mechanism of the reaction would be of the "ping pong" type. In the case of the E G F receptor kinase it has been shown that the enzyme follows an ordered sequential mechanism and not a ping pong mechanism (69). This suggests that

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at least in the case of the E G F receptor kinase system the phosphorylated enzyme is not a normal phosphoenzyme intermediate of the phosphotransferase reaction. Why do tyrosine protein kinases phosphorylate themselves? Does it represent an important regulatory step or a side reaction of little significance? In the case of the insulin-stimulated protein kinase some experimental evidence is available to suggest that phosphorylation of the receptor subunit activates the tyrosine protein kinase (19). Such an activation of the tyrosine protein kinase upon autophosphorylation has not been shown with other protein kinases. This possibility needs to be explored experimentally. Recently, G o r d o n et al. (102) have reported that incubation of RSVtransformed chicken embryo fibroblasts with 1-100 #M vanadate results in increased phosphorylation of pp60 ~rc as well as increased protein kinase activity of pp61Yre. Whether the increased protein kinase activity of pp60 ~rc is due to an increased phosphorylation or vice versa is a matter of speculation at present. Using site-specific mutagenesis, a mutant of RSV has been constructed in which a tyrosine residue at position 416 of pp60 v.... (the major site of phosphorylation at tyrosine) has been replaced by phenylalanine (103). This substitution has no effect on the tyrosine protein kinase activity of pp60 v.... and the protein is still capable of cell transformation. Whether this substitution has any effect on kinetic properties of the protein kinase activity of mutant pp60 v.... has not been determined. Cells transformed by this mutant virus, like wild type, show elevated levels of phosphotyrosine in cellular proteins. However, some other Tyr sites in mutant pp60 v.... are still phosphorylated and they perhaps are also phosphorylated in wild type pp60 v-src. The role of phosphorylation of these sites is not known. Divalent m e t a l ion requirement. Tyrosine protein kinases require Mg 2÷ or Mn 2÷ as divalent cations for the expression of their catalytic activity. In many cases the concentration of Mg2÷ (20-50 mM) or Mn 2÷ (5-10 mM) required for maximal kinase activity is very high; this suggests that metal ion may serve as an activator in addition to its requirement for the formation of M g A T P (or MnATP) (10). Some of the tyrosine protein kinases apparently show higher activity with Mn 2÷ than with Mg 2÷ (Table 1 and 2). However, in many of these studies the concentration of ATP (or GTP) used was very low (I 0 #M or lower) and the phosphorylation was measured after a given time of incubation. At low A T P concentrations in the presence of Mn 2÷ apparently higher levels of phosphorylation may be observed if the Km of the kinase for M n A T P is lower than that of MgATP. Secondly Mr~ ÷ appears to inhibit particulate tyrosine specific protein phosphatase activity (23, G~. Swarup and D. L. Garbers, unpublished observation). In any case it seems unlikely that Mn 2÷ can be the physiological cation since the concentrations of Mn 2÷ in vivo are very low (0.2-1.0 #M in isolated rat hepatocytes) (104, 105).

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Regulators of tyrosine protein kinases. EGF, insulin, P D G F and perhaps some transforming growth factors (9, 19-22, 106, 107) appear to be regulators of tyrosine protein kinase activity. At present no regulator is known for viral tyrosine protein kinases with the possible exception of pp6tYrc. The tyrosine protein kinase activity of pp6lYrc towards vinculin as substrate (but not casein) is greatly stimulated by the presence of certain phospholipids like phosphatidylglycerol and phosphatidyl serine, but not by phosphatidylcholine 008). The interconversion of phospholipids may therefore influence the substrate specificity of the pp60 ~rc kinase, since most tyrosine protein kinases are associated with membranes where phospholipids are potential regulators. Cyclic-AMP dependent protein kinase is known to phosphorylate a serine residue of pp6tYrc (109). Whether this phosphorylation results in altered kinase activity is not known. Most of the tyrosine protein kinases (all viral kinases, E G F receptor, insulin receptor) are known to be phosphorylated in vivo at serine residues in addition to phosphorylation at a tyrosine residue. No reports yet exist which identify the protein kinases involved or the significance of such phosphorylations. Phosphorylation of IgG. The protein products of viral oncogenes coding for tyrosine protein kinases were identified by the use of antisera directed against the gag protein or the onc gene products. When these immunoprecipitates containing viral onc gene products were incubated with [7-32p]ATP, the heavy chain of IgG was phosphorylated at a tyrosine residue except in the case of v-abl (Table I). The antibodies present in TBR serum (containing IgG directed against pp6lYr0 were also phosphorylated by the E G F receptor kinase, insulin-stimulated kinase and by the tyrosine protein kinase from the particulate fraction of rat spleen (shown here). These three protein kinases were not precipitated by TBR serum. The significance of these phosphorylations is a matter of speculation. Does the phosphorylation of IgG heavy chain of TBR serum by these kinases represent only the phosphorylation of an exogenous, non-specific substrate like casein or histones? Do these protein kinases which phosphorylate IgG heavy chain of TBR serum have a common or closely related general structure or catalytic site? Comparison of amino acid sequences (derived mostly from DNA sequence analysis) of transforming proteins coded by viral abl, src,fes, yes andfps genes has revealed that these proteins have extensive sequence homology (111). The carboxy terminus regions of these proteins which are implicated in the active site for tyrosine phosphorylation show more pronounced homology (111). In addition pp60 v.... shows amino acid sequence homology with the catalytic subunit of bovine cyclic AMP dependent protein kinase, especially in the carboxyl terminus region where the protein kinase activity is localized (112). A region of 104 contiguous amino acids in P D G F is virtually identical with the putative transforming protein p28 sis of simian sarcoma virus (113, 114).

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C o n s i d e r i n g these relationships it w o u l d not be surprising if one finds that the catalytic sites o f pp60 v~rc, E G F r e c e p t o r p r o t e i n kinase, insulin s t i m u l a b l e p r o t e i n kinase a n d the m a j o r tyrosine p r o t e i n kinase f r o m rat spleen are r e l a t e d f u n c t i o n a l l y a n d p e r h a p s structurally.

SUMMARY Tyrosine-specific p r o t e i n kinases f r o m n o r m a l tissue have been studied using synthetic peptides as substrate. Spleen h a d m u c h higher activity o f the enzyme in the p a r t i c u l a t e fraction t h a n any o t h e r n o r m a l tissue (except purified T lymphocytes). The tyrosine p r o t e i n kinase f r o m the p a r t i c u l a t e fraction of rat spleen was p a r t i a l l y purified a n d characterized. T h e kinase c o u l d p h o s p h o r y l a t e src-related as well as u n r e l a t e d peptides a n d casein at tyrosine residues. T h e enzyme in the m e m b r a n e seemed to have s o m e w h a t different s u b s t r a t e specificity t h a n the solubilized, p a r t i a l l y purified enzyme. S e r u m c o n t a i n i n g a n t i b o d y to pp60 v.... d i d n o t precipitate the kinase; however, the p r o t e i n kinase could p h o s p h o r y l a t e the heavy chain o f l g G from T B R serum (but not from n o r m a l serum). T h e possible r e l a t i o n s h i p o f the tyrosine-specific p r o t e i n kinase o f spleen with pp6ff .... a n d o t h e r tyrosinespecific p r o t e i n kinases is discussed.

ACKNOWLEDGEMENTS W e w o u l d like to t h a n k Mr. A m r i k K h a t r a for his excellent technical assistance a n d D i a n e S m i t h s o n for t y p i n g this m a n u s c r i p t .

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