©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Extraordinary Active Site Substrate Specificity of pp60
A MULTIPLE SPECIFICITY PROTEIN KINASE (*)

(Received for publication, November 9, 1994)

Tae Ryong Lee Jinkui Niu David S. Lawrence (§)

From the Departments of Chemistry and Medicinal Chemistry, Natural Sciences and Mathematics Complex, State University of New York, Buffalo, New York 14260

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We report the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60. Like the cAMP-dependent protein kinase and protein kinase C, pp60 will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60 phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60 is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S.(1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60 does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60 phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60 is discussed. Finally, the active site specificity of pp60 is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well.


INTRODUCTION

The vast majority of enzymes that participate in growth-promoting signal transduction pathways are protein kinases(1, 2) . Furthermore, the link between carcinogenesis and certain constituitively activated pathways has been clearly established(1, 2) . Consequently, it is not surprising that there is a keen interest in the creation of protein kinase inhibitors. However, in order for these inhibitors to be effective they must be designed with specific criteria in mind. First, the structural attributes of the inhibitory species must complement those of the target enzyme. For example, if the mechanism of inactivation is one that requires interaction with the enzyme active site, then the inhibitory component must be readily accommodated and properly positioned within the active site region. Second, protein kinase inhibitors will only prove useful if they can be targeted against specific kinases. This is a potentially difficult challenge, since the reaction catalyzed by protein kinases, namely phosphoryl transfer from ATP to an acceptor hydroxyl moiety in proteins, is a relatively general one. Fortunately, most protein kinases will utilize short peptides as substrates, and it is now apparent that substrate recognition is often driven by the primary sequence associated with the peptide undergoing phosphorylation(3) . In short, it may be possible to target inhibitors for specific protein kinases by utilizing peptides containing the appropriate primary sequence. Unfortunately, many protein kinases exhibit overlapping sequence specificities, thereby limiting the universal applicability of this approach.

Recently, we developed a facile method to rapidly assess the active site specificity of protein kinases(4) . We have found that protein kinases will phosphorylate a diverse array of alcohol-containing non-amino acid residues(4, 5, 6, 7, 8, 9) . Most importantly, this approach allows us to address the specific issues noted above concerning protein kinase inhibitor design. In particular, the range of alcohol-bearing compounds that serve as protein kinase substrates should provide a good assessment of the range of functionality that can be readily accommodated within the active site region. Second, we have recently shown that even those protein kinases that exhibit overlapping substrate specificities for conventional peptides will display remarkably distinct specificities for peptides bearing unconventional alcohol-containing residues(8) . In short, the active site substrate specificity of protein kinases, coupled with their characteristic sequence specificities, may ultimately provide a useful method for specifically targeting protein kinases in the cell. Up until this point, our work has dealt exclusively with serine/threonine-specific protein kinases. However, in this paper we describe the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60. Much to our surprise, we have found that the specificity of pp60 is extraordinarily broad and includes a unusually diverse structural array of alcohol-containing residues.


MATERIALS AND METHODS

All chemicals were obtained from Aldrich, except for [-P] ATP (DuPont NEN), piperidine (Advanced ChemTech), protected amino acid derivatives (Advanced Chemtech and Bachem), Liquiscint (National Diagnostics), and the Fmoc(^1)-Leu-2-methoxy-4-alkoxybenzyl alcohol resin (Peninsula Laboratories, Inc.). Phosphocellulose P-81 paper disks were purchased from Whatman. ^1H NMR and C NMR experiments were performed at 400 MHz (Varian VXR-400S) and 22.5 MHz (Varian Gemini-300), respectively. Chemical shifts are reported with respect to tetramethylsilane. Fast atom bombardment (peptides) and chemical ionization (aminoalcohols) mass spectral analyses were conducted with a VG-70SE mass spectrometer.

Aminoalcohol Syntheses

All aminoalcohol derivatives were purchased from Aldrich, except for (4-aminomethyl)benzyl alcohol, (3-aminomethyl)benzyl alcohol, 7-amino-1-heptanol, 8-amino-1-octanol, 10-amino-1-decanol, and 12-amino-1dodecanol.

Protocol for the Synthesis of (3-Aminomethyl)benzyl Alcohol, (4-Aminomethyl)benzyl Alcohol, 7-Amino-1-heptanol, 8-Amino-1-octanol

3-Cyanobenzaldehyde (0.5 g, 3.8 mmol) was slowly added to a refluxing solution of suspended LiAlH(4) (1.0 g, 26.3 mmol) with vigorous stirring in anhydrous ethyl ether (100 ml) maintained under an argon atmosphere. The solution was heated at reflux for 10 h and then water was slowly added dropwise to quench the reaction (under no further evolution of H(2) was apparent). The solidified reaction mixture was vigorously stirred with a 1:1 mixture of CHCl(3)/ethyl ether (3 times 80 ml), and the combined extract was dried over Na(2)SO(4) and filtered. Evaporation of the solvent furnished a slightly yellow residue which was purified on a silica gel column (100-200 mesh), employing a 70:25:5 CHCl(3):CH(3)OH:NH(3)/H(2)O solvent. Upon solvent evaporation, (3-aminomethyl)benzyl alcohol was obtained as a white solid in 67% yield. ^1H NMR (Me(2)SO-d(6)): 7.09-7.23 (m, 4H, aromatic hydrogens); 4.44 (s, 2H, -CH(2)-OH); 3.65 (s, 2H, -CH(2)-NH(2)). C NMR (Me(2)SO-d(6)): 144.4, 142.8, 128.2, 125.7, 125.6, and 124.7 (aromatic C); 63.2 (-CH(2)-OH); 45.8 (-CH(2)-NH(2)). CI-MS m/e calculated for C(8)HNO: 137.2; found: 138.3 (M). (4-aminomethyl)benzyl alcohol was obtained from methyl 4-cyanobenzoate using the above protocol in 76% yield. ^1H NMR (Me(2)SO-d(6)): 7.24 (q, 4H, aromatic hydrogens); 4.44 (s, 2H, -CH(2)-OH); 3.67 (s, 2H, -CH(2)-NH(2)). C NMR (Me(2)SO-d(6)): 142.9, 140.7, 127.0, 126.6 (aromatic C); 62.8 (-CH(2)-OH); 45.4 (-CH(2)-NH(2)). CI-MS m/e calculated for C(8)HNO: 137.2; found: 138.1 (M). 7-Amino-1-heptanol was obtained from 7-aminoheptanoic acid using the above protocol in 64% yield. ^1H NMR (CDCl(3)): 3.59 (t, 2H, -CH(2)-OH); 2.64 (t, 2H, -CH(2)-NH(2)); 1.30-1.51 (m, 10H, remaining aliphatic hydrogens). C NMR (CDCl(3)): 63.2 (-CH(2)-OH); 42.4 (-CH(2)-NH(2)); 33.9, 33.0, 29.5, 27.0, 25.9 (remaining aliphatic carbons). CI-MS m/e calculated for C(7)HNO: 131.3; found: 132.2 (M). 8-Amino-1-octanol was obtained from 8-aminooctanoic acid using the above protocol in 55% yield. ^1H NMR (CDCl(3)): 3.58 (t, 2H, -CH(2)-OH); 2.64 (t, 2H, -CH(2)-NH(2)); 1.28-1.52 (m, 12H, remaining aliphatic hydrogens). C NMR (CDCl(3)): 63.2 (-CH(2)-OH); 42.4 (-CH(2)-NH(2)); 33.9, 33.0, 29.63, 29.59, 27.0, 25.9 (remaining aliphatic carbons). CI-MS m/e calculated for C(8)HNO: 145.3; found: 146.2 (M).

Protocol for the Synthesis of 10-Amino-1-decanol and 12-Amino-1-dodecanol

A solution of potassium phthalimide (0.65 g, 3.5 mmol) and 10-bromo-1-decanol (0.71 g, 3 mmol) in DMF (10 ml) was heated to reflux for 4 h. The reaction was allowed to cool and was then filtered to remove the white precipitate, and the solvent was subsequently removed in vacuo. The residue obtained was then dissolved in 30 ml of ethanol containing 0.5 ml of 98% hydrazine (16 mmol). The mixture was heated to reflux for 4 h, cooled to room temperature, and filtered to remove the precipitate (phthalhydrazide). This precipitate was washed three times with chloroform (20 ml) and the combined extracts added to the ethanol filtrate. A white solid was obtained after evaporating the solvent in vacuo. The solid was further purified via column chromatography on silica gel (100-200 mesh; 70:25:5 CHCl(3):CH(3)OH:NH(3)/H(2)O) to furnish 10-amino-1-dodecanol in 85% yield. ^1H NMR (CDCl(3)): 3.60 (t, 2H, -CH(2)-OH); 2.65 (t, 2H, -CH(2)-NH(2)); 1.25-1.52 (m, 16H, remaining aliphatic hydrogens). C NMR (CDCl(3)): 63.4 (-CH(2)-OH); 42.4 (-CH(2)-NH(2)); 33.7, 33.0, 29.7 (two peaks), 29.61, 29.57, 27.0, 25.9 (remaining aliphatic carbons). CI-MS m/e calculated for CHNO: 173.4; found: 174.4 (M). 12-Amino-1-dodecanol was obtained from 12-bromo-1-dodecanol using the above protocol in 91% yield. ^1H NMR (CDCl(3)): 3.59 (t, 2H, -CH(2)-OH); 2.64 (t, 2H, -CH(2)-NH(2)); 1.23-1.52 (m, 20H, remaining aliphatic hydrogens). C NMR (CDCl(3)): 63.3 (-CH(2)-OH); 42.3 (-CH(2)-NH(2)); 33.7, 33.0, 29.75 (two peaks), 29.71 (two peaks), 29.62, 29.58, 27.0, 25.9 (remaining aliphatic carbons). CI-MS m/e calculated for CHNO: 201.5; found: 202.5 (M).

Peptide Synthesis

Fmoc-Arg[4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr)]-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-LeuLeu-resin was prepared on the 2-methoxy-4-alkoxybenzyl alcohol resin (substitution level = 0.45 mmol/g of resin) with fluorenylmethoxycarbonyl (Fmoc) amino acids(10, 11) . A standard Fmoc peptide synthesis protocol was employed using an automated peptide synthesizer (Advanced ChemTech Act 90). Upon completion of peptide synthesis, the side chain-protected peptide was cleaved from the resin using 1% trifluoroacetic acid in CH(2)Cl(2) (20 ml/g of resin; 15 min). The resin was filtered and the trifluoroacetic acid/CH(2)Cl(2) solvent (containing the peptide) was collected. This process was repeated three additional times. 100 ml of water was subsequently added to the combined trifluoroacetic acid/CH(2)Cl(2) extracts. Triethylamine was added to furnish a neutral solution and then all but 15 ml of the solvent was removed under reduced pressure. The precipitated peptide was collected via filtration and washed with ethyl ether (50 ml). Typically, 1 g of resin provided 400 mg of side chain-protected peptide. The free C terminus of FmocArg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-Leu-Leu (40 mg, 0.014 mmol) was activated via treatment with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (Bop; 0.028 mmol) in 3 ml of CH(2)Cl(2)/DMF (1:1) for 3 min. Individual amino alcohols (0.14 mmol) were introduced and allowed to react with the activated peptide for 1 h. Subsequently, the N-terminal Fmoc protecting group was removed via treatment with piperidine for 30 min, and the N terminus deprotected peptide alcohol was then precipitated from solution by addition of ethyl ether (50 ml). The heterogeneous solution was filtered in order to isolate the peptide. The remaining protecting groups (Mtr and t-butyl ester) were removed by dissolving the peptide alcohol in 90% trifluoroacetic acid, 10% thioanisol (6 ml). After stirring for 6 h, the peptide was precipitated from solution (50 ml of ethyl ether) and subsequently isolated via filtration. Each peptide alcohol was then purified via a two-step sequence. First, the individual peptides were ion-exchanged on CM-Sephadex C-25 (0.4 M KCl to a 1.2 M KCl gradient using a 50 mM potassium acetate, pH 3.5, buffer). The desired peptide alcohol typically eluted at 0.8-1.0 M KCl was subsequently collected and lyophilized. The peptides were then desalted via preparative HPLC using three Waters radial compression modules (2.5 times 10 cm) connected in series (gradient (solvent A: 0.1% trifluoroacetic acid in water; solvent B: 0.1% trifluoroacetic acid in acetonitrile): 0-3 min (100% A); a linear gradient from 3 min (100% A) to 5 min (80% A and 20% B); 5-20 min (20% B to 50% B); 20-25 min (50% B to 80% B). All of the purified peptides gave satisfactory fast atom bombardment mass spectral analyses.

Peptides 1-5 were prepared as C terminus amides on the benzhydrylamine resin with t-butyloxycarbonyl (t-Boc) amino acids employing a previously described protocol(12) . The peptides were simultaneously deprotected and cleaved from the resin by sequential treatment with 50% methyl sulfide, 50% HF (30 min) and 10% anisole, 90% HF (45 min). All of the crude peptides were purified by cation-exchange chromatography on CM-Sephadex C-25, followed by preparative HPLC as described above.

Peptides 21 and 26 were synthesized by first coupling Fmoc-protected L-serine and Fmoc-protected D-tyrosine, respectively, to the rink resin(13) . The Fmoc protecting groups were then removed with piperidine. The free C terminus of Fmoc-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-Leu-Leu (40 mg, 0.014 mmol; see synthesis described above) was activated with Bop (0.028 mmol) in 10 ml of CH(2)Cl(2)/DMF (1:1). The amino acid-substituted rink resins (0.042 mmol) were then added to separate solutions containing the activated form of Fmoc-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Arg(Mtr)-Leu-Glu(t-butyl)-Glu(t-butyl)-Leu-Leu. The Fmoc group was removed (piperidine) and the undecapeptides cleaved (90% trifluoroacetic acid, 10% thioanisole; 10 ml, 4 h) from the rink resin. The peptides were isolated and purified as described above. Both purified peptides gave satisfactory fast atom bombardment mass spectral analyses.

Human Recombinant pp60

Human pp60 was purchased from Upstate Biotechnology Inc. The enzyme was expressed by recombinant baculovirus containing the human c-src gene in SF9 insect cells.

Kinase Assay

Assays were performed in triplicate at pH 7.5 and thermostatted in a water bath maintained at 22 °C. Final assay volume totaled 40 µl and contained 20 mM Hepes, 20 mM MgCl(2), 0.125 mg/ml bovine serum albumin, 100 µM (Na(3)VO(4)), and 1.55 nM pp60 (except for peptides 8 and 9, where [pp60] = 0.775 nM). For the determination of kinetic constants the following concentrations were employed: 100 µM [-P]ATP (1000-2000 cpm/pmol) and a substrate concentration over a 10-fold range around the apparent K(m). Phosphorylation reactions were initiated by addition of 10 µl of pp60 diluted from a concentrated stock solution (6.2 nM (or where appropriate, 3.1 nM) in 1 mM dithiothreitol and 20 mM Hepes, pH 7.5) to a solution containing 15 µl of peptide (20 mM Hepes, pH 7.5) and 15 µl of assay buffer (20 mM Hepes, 0.33 mg/ml bovine serum albumin, 53.33 mM MgCl(2), and 233 µM Na(3)VO(4)). Reactions were terminated after 20.0 min by spotting 25-µl aliquots onto 2.1-cm diameter phosphocellulose paper disks. After 10 s the disks were immersed in 10% glacial acetic acid and allowed to soak with occasional stirring for at least 1 h. The acetic acid was decanted, and the disks were collectively washed with 4 volumes of 0.5% H(3)PO(4), 1 volume of water, followed by a final acetone rinse. The disks were air-dried and placed in plastic scintillation vials containing 6 ml of Liquiscint prior to scintillation counting for radioactivity.

Determination of Kinetic Constants

The apparent K(m) (± S.D.) and V(max) (± S.D.) values were determined from initial rate experiments. The data from these experiments were plotted using the Lineweaver-Burk procedure, and the corresponding plots proved to be linear.


RESULTS AND DISCUSSION

We have recently shown that the active site substrate specificity of both the cAMP-dependent protein kinase (``PKA'') as well as protein kinase C (``PKC'') is not limited to serine and threonine moieties(4, 5, 6, 7, 8, 9) . Indeed, these protein kinases will phosphorylate a variety of non-amino acid residues, if these residues are properly positioned on active site-directed peptides. This has allowed us to assess which structural features can (and cannot) be readily tolerated within the active site of these enzymes. In addition, we were surprised to find that the active site specificity of PKA and PKC dramatically differ with respect to non-amino acid residues(8) . This is in spite of the fact that both of these protein kinases exhibit overlapping substrate specificities with conventional synthetic peptides. These results suggest that protein kinases can be distinguished from one another on the basis of their active site specificities, even if their sequence specificities are nearly identical. We have now applied this analysis to a tyrosine-specific protein kinase. The range of structurally diverse species that are phosphorylated by pp60 is extraordinarily broad, much more so than that of PKC and PKA. Consequently, the remarkably accommodating nature of the pp60 active site should afford considerable flexibility in terms of inhibitor design.

We have previously noted that an active site specificity analysis requires ready access to a family of peptides containing structurally dissimilar residues at the site of phosphorylation(4) . In the majority of protein kinase peptide substrates that have been described to date, the phosphorylatable residue typically resides at a nonterminal position within the peptide(3) . Unfortunately, the preparation of peptides containing exotic residues at internal positions can be synthetically challenging, particularly if the residue of interest contains hypersensitive functionality that would not ordinarily survive the harsh conditions of solid phase peptide synthesis. In addition, even if peptides of this sort could be readily prepared, for every peptide-based substrate candidate to be investigated a complete peptide would have to be synthesized. These time-consuming synthetic obstacles can be circumvented if the alcohol-bearing residue can be directly attached to the intact peptide at either the N or C terminus. Under these circumstances, the key residue is not exposed to the acidic and/or basic conditions associated with peptide synthesis, and in addition, the intact peptide precursor need be synthesized only once. However, in order for this type of approach to be successful, the target protein kinase must be able to phosphorylate residues at a terminal position in synthetic peptides. Indeed, we have recently identified a number of potent substrates for various protein kinases (e.g. PKA, PKC, cdc2^2, CaM-kinase Ia(^3)) in which a serine or threonine residue is positioned at the C or N terminus. Furthermore, Pinna and his colleagues have recently shown that several tyrosine-specific protein kinases (members of the src kinase family) isolated from spleen will phosphorylate peptides containing tyrosine located at either the N or C terminus(14) . Consequently, it seemed likely that pp60 itself would exhibit analogous behavior.

We examined the relative rates of phosphorylation of peptides 1-5 by pp60. Previous work by others have shown that acidic residues can be important determinants for substrate recognition by pp60(14, 15, 16) . In addition, since we chose to utilize the phosphocellulose binding assay, we attached multiple arginine residues to these peptides in order to promote efficient coordination of the phosphorylated product to the phosphocellulose disc. On the basis of the results in Table 1we decided to employ peptide 5 as the parent substrate. (^4)



We previously prepared peptides containing alcohol-bearing residues situated at the C terminus by utilizing an oxime resin for solid phase peptide synthesis(4) . Unfortunately, we have found that this synthetic strategy leads to by-products as a consequence of the reactivity of the protected glutamic acid moieties contained within the peptide. (^5)Consequently, all of the peptide alcohols described herein were prepared via activation of the protected peptide 6 with the condensing agent 7 (Fig. 1). The desired peptide alcohols were obtained upon treatment of the activated form of 6 with various amino alcohols (followed by sequential deprotection with piperidine and trifluoroacetic acid).


Figure 1: Synthesis of the peptide-based pp60 substrates. Fmoc-protected amino acids were employed during solid phase peptide synthesis. The synthetic peptide was cleaved from the 2-methoxy-4-alkoxybenzyl alcohol resin with side chain-protecting groups intact using 1% trifluoroacetic acid in CH(2)Cl(2). The protected peptide was then coupled to the desired aminoalcohols listed using the Bop (7) activating agent. Deprotection furnished the peptides 9-25. See ``Materials and Methods'' for the preparation of peptides 2 and 26.



We note the following concerning the active site substrate specificity of pp60.

Achiral Aromatic and Benzylic Alcohols Serve as pp60 Substrates

We have previously demonstrated that both PKA and PKC will phosphorylate achiral residues (8) . In an analogous fashion, pp60 will not only phosphorylate the aromatic alcohol of tyrosine, but the hydroxyl moiety of achiral residues as well. For example, the tyramine-containing peptide 9 exhibits a somewhat better K(m) than that of the parent peptide 8 (Table 2). This behavior is in marked contrast to that displayed by PKA and PKC, where peptide substrates containing achiral residues exhibit elevated K(m) values relative to their chiral amino acid counterparts(8) . We have also previously shown that PKA will phosphorylate both aliphatic and aromatic alcohols(4, 9) . Consequently, we investigated the ability of pp60 to catalyze the phosphorylation of the benzylic alcohol-containing peptides 10 and 11. Both peptides serve as substrates, with species 10 exhibiting a relatively impressive K(m) but a modest V(max). There are several noteworthy features concerning these results with respect to inhibitor design. Inhibitors, particularly transition state analogs or suicide substrates, often contain elaborate functionality whose preparation can be time-consuming. The latter is especially true if the key functionality of interest must be incorporated onto a chiral residue. The fact that pp60 will recognize and catalyze the phosphorylation of structurally simple achiral alcohols should simplify the synthesis of novel inhibitory species. In addition, the active site of pp60 appears to be particularly accommodating since the enzyme phosphorylates both aromatic and aliphatic alcohols, including the two structurally dissimilar benzylic alcohols 10 and 11.



Straight Chain Achiral Aliphatic Alcohols Serve as pp60 Substrates

PKA will not catalyze the phosphorylation of tyrosine(7) . However, this ``serine/threonine''-specific kinase will phosphorylate aromatic alcohols as long as the hydroxyl moiety on these residues can be positioned in the active site in a manner comparable with that of serine or threonine. In an analogous fashion, one might predict that if an aliphatic side chain can properly orient a hydroxyl functionality into the active site of a tyrosine-specific protein kinase, then phosphoryl transfer should ensue as well. To a rough approximation, this corresponds to a residue containing a side chain length of six carbon atoms (see Fig. 2). Therefore, one might expect that chain lengths much smaller or larger than this ideal would not be able to optimally position the phosphoryl acceptor hydroxyl moiety in the appropriate region of the enzyme active site. In spite of this eminently reasonable assumption, it is clear that pp60 does not discriminate in the expected fashion on the basis of chain length (Table 3). For example, pp60 phosphorylates peptide 12, in spite of the fact that the distance from the hydroxyl moiety to the peptide backbone is identical to that present in serine and significantly shorter than that in tyrosine. Furthermore, there is little variance in k/K(m) from the two carbon-bearing alcohol (i.e.12) up to the ten carbon-containing species (i.e.19). Indeed, only with peptide 20, which possesses a side chain of 12 carbon atoms, is there a significant loss in substrate effectiveness. These observations are certainly not in accord with those from our earlier analysis of the substrate specificity of PKA and PKC. We previously found that both enzymes will phosphorylate a variety of alcohol-containing residues, as long as the hydroxyl moiety in these substrates is positioned the appropriate distance from the peptide backbone (i.e. the same as that present in serine and threonine)(8) . For example, a dramatic reduction in substrate efficacy is observed if the side chain is altered from the ideal length by as little as two carbon atoms. Furthermore, this dependence upon side chain length is not just limited to the activity of PKA and PKC. For example, Barford et al. (17) have recently proposed that, on the basis of the three-dimensional structure of protein tyrosine phosphatase 1B, the depth of the active site plays a critical role in enabling tyrosine-specific protein phosphatases to distinguish between target phosphotyrosine residues and those of phosphoserine and phosphothreonine. Clearly, pp60 does not conform to the limitations displayed by these related enzymatic species. What is the structural basis for this behavior? In addition, if the tyrosine-specific pp60 is able to phosphorylate 12, shouldn't it be able to phosphorylate serine as well?


Figure 2: Comparison of the side chain lengths of the tyrosine- and 6-amino-1-hexanol-containing peptides.





An L-Serine-containing Peptide Serves as a Substrate for pp60

Since ethanolamine-bearing peptide 12 is a substrate for pp60, we investigated the possibility that the corresponding serine-containing peptide 21 could also serve as a substrate for this ``tyrosine-specific'' protein kinase. Indeed, the latter is a substrate (Table 4), albeit a significantly poorer one than the former. Nevertheless, this observation is clearly reminiscent of the behavior of a small group of protein kinases; namely those that are apparently able to phosphorylate serine, threonine, and tyrosine(18) . What is the structural basis for this dual specificity? One possibility is that the active site is inherently flexible and is able to assume a conformation that structurally complements that of the active site-bound residue. However, this does not account for the difference in k/K(m) values between peptides 12 and 21. An alternative explanation for this ``dual specificity'' assumes a more conventional conformationally fixed active site. In the model illustrated in Fig. 3, the peptide is able to occupy any of several closely positioned loci on the enzyme surface, each of which lies a characteristic distance from the phosphorylation site. In this scenario, an alcohol moiety positioned on a ``shorter-than-ideal'' side chain (e.g.12) can assume the proper location in the active site if a portion of the peptide backbone can be accommodated in the active site region. For an alcohol functionality located on a ``longer-than-ideal'' side chain (e.g.20), the peptide backbone would be positioned at a greater distance from the active site. Implicit within this model is the assumption that the interaction between the peptide substrate and the enzyme surface is not fixed to a single prescribed site, but instead fluctuates according to the structural nature of the substrate. For example, this behavior might be observed if the enzyme surface is characterized by an extended region of positive charge density. Under these circumstances, the peptide could favorably associate with the enzyme at a multitude of sites. In addition, this model may offer an explanation for the disparate K(m) values associated with the phosphorylation of peptides 12 and 21. While incorporation of the alcohol moiety of 12 into the active site should not be structurally demanding, insertion of the hydroxyl functionality of serine (i.e.21) by necessity, positions a structurally obtrusive amide group in the active site. If this notion is correct, then a residue that contains a substituent even larger than the amide moiety should produce a poorer substrate. Indeed, peptide 22, which possesses a benzyl substituent at the alpha-position, is such a poor substrate that we were unable to obtain accurate kinetic constants. We also examined the ability of peptide 24, which contains a methyl substituent at the beta position, to serve as a substrate for pp60. This peptide proved to be an exceedingly poor substrate as well. Finally, we prepared the diastereomers of 22 (i.e.23) and 24 (i.e.25). These peptides also failed to serve as efficient substrates. In short, it is clear that substituents at the alpha or beta positions of the phosphorylatable residue dramatically interfere with the ability of short chain alcohols to serve as a substrates for pp60. While these observations are certainly consistent with the model depicted in Fig. 3, it remains to be seen whether this type of analysis is applicable to the several dual specificity protein kinases that have recently been identified(18) .




Figure 3: Interactions of alcohol-containing peptides with pp60. A, the tyrosine-bearing peptide 8 is aligned on the enzyme surface to properly position the aromatic alcohol in the active site. B, since the ethyl side chain of 12 is short relative to that in 8, the P-1 residue must be partially inserted into the active site in order for the hydroxyl functionality to be correctly situated in the active site. C, the side chain of 20 is of sufficient length so that the peptide backbone may be located at a greater distance from the active site than that in B. D, unfavorable steric interactions between active site residues and the alpha-amide moiety of L-serine may arise upon interaction of 21 with pp60.



pp60 Phosphorylates a D-Tyrosine Residue in an Active Site-directed Peptide

Although it is clear that substituents at either the alpha or beta positions in the ethylalcohol-based species (12) markedly lower substrate efficacy (cf. 21-25), this is obviously not the case with the aromatic alcohol-based peptides 8 and 9. Indeed, the alpha-amide substituent in 8 is actually beneficial in terms of k/K(m). Evidently, alpha-substituents can be accommodated near the active site as long as these substituents are positioned some distance from the alcohol that suffers phosphorylation. This is also consistent with the model depicted in Fig. 3. While it is clear that an alpha-substituent can have favorable consequences in terms of substrate efficacy for aromatic alcohols (e.g.8), we wondered whether the configuration at the alpha-center played any role in substrate recognition. We(8) , as well as others(19, 20) , have shown that PKC will catalyze the phosphorylation of both D- and L-residues, whereas the specificity of PKA is strictly limited to L-residues. Indeed, the D-tyrosine-containing peptide 26 does serve as a substrate for pp60, although not as efficiently as its' isomeric counterpart 8. We have not yet conclusively resolved how a D-amino acid residue can be recognized by the active sites of PKC and pp60. In particular, if the active site regions of these enzymes are analogous to that of PKA, then the Michaelis complex of a D-amino acid residue should experience severe steric interactions that would preclude the requisite alignment of the hydroxyl moiety in the active site(8) . However, we have previously proposed that the alcohol functionality in a D-residue could be presented to the active site of PKC in a structurally acceptable fashion if the peptide is able to bind to the enzyme surface in a retrograde C to N terminus sense(8) . An analogous binding mode may be in operation for pp60 as well.

In summary, pp60 exhibits an unusually broad active site substrate specificity. This protein kinase is able to catalyze the phosphorylation of achiral residues, including aromatic and aliphatic alcohols. Furthermore, whereas the substrate specificity of PKA and PKC is limited to straight chain aliphatic alcohols of four carbons or less, pp60 phosphorylates aliphatic alcohols that vary from two to twelve carbon atoms in length. Although, short chain aliphatic alcohols containing substituents at either the alpha and beta positions are extraordinarily poor substrates, aromatic alcohols containing alpha-substituents are superior substrates (cf. 8 and 9). Most interestingly, the configuration at the alpha position is not a critical element in substrate recognition. Peptides containing either D- or L-tyrosine serve as pp60 substrates. These observations may very well have profound implications in terms of inhibitor design. For example, it should be possible to synthesize inhibitors of pp60 from readily available compounds, since this enzyme recognizes and phosphorylates simple achiral residues. In addition, since pp60 is able to utilize a variety of structural variants as substrates, the potential flexibility associated with inhibitor design should be equally as great. Finally, since the synthetic approach employed in this study does not expose key residues to the conditions of solid phase peptide synthesis, elaborate functionality that might not ordinarily survive solid phase synthesis can be easily incorporated into the active site-directed peptide.


FOOTNOTES

*
This work was supported by a grant from the United States Army Medical Research and Development Command. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 716-645-6800 (ext. 2170); Fax: 716-645-6963.

(^1)
The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; Boc, t-butyloxycarbonyl; Bop, benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate; Mtr, 4-methoxy-2,3,6-trimethylbenzenesulfonyl; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; HPLC, high performance liquid chromatography; DMF, N,N-dimethylformamide; CI-MS, chemical ionization mass spectrometry.

(^2)
J. Srinivasan, M. Koszelak, M. Mendelow, Y.-G. Kwon, and D. S. Lawrence, unpublished results.

(^3)
J. C. Lee, Y.-G. Kwon, D. S. Lawrence, and A. M. Edelman, unpublished results.

(^4)
Peptide 5 does differ from the parent peptide 8 in one respect, namely a leucine-for-alanine exchange at the P-5 position. We decided to incorporate leucine at this position in the final peptide to reduce the number of different amino acids contained within the active site-directed peptide.

(^5)
Benzyl ester-protecting groups are employed on the side chains of the glutamic acid residues for syntheses on the oxime resin (which utilized the Boc protection strategy). We found that these protecting groups are prone to displacement by amines, thereby leading to undesired amide by-products.


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