An early method for assaying cAMP levels in crude extracts of skeletal muscle led to the discovery of a heat-stable inhibitor protein of cAMP-induced phosphorylation(1) . This protein kinase inhibitor (PKI) ()was eventually purified (2) and shown to be a small, specific(3) , and potent (K = 0.2 nM) competitive inhibitor of the catalytic (C) subunit of cAMP-dependent protein kinase(4, 5) .

A variety of peptide studies have established the significance of certain PKI amino acids in the inhibition of C subunit by PKI(6, 7) . Two such residues, Arg and Arg, are located in the amino-terminal inhibitory region of PKI as part of a pseudosubstrate site (Arg-Arg-Asn-Ala) that mimics the Arg-Arg-X-(Ser or Thr) consensus sequence for phosphorylation of substrates by cAMP-dependent protein kinase(8, 9) . While these pseudosubstrate arginines are necessary for inhibition of C subunit by PKI peptides, their presence is not sufficient to explain the subnanomolar K of native PKI. Indeed, inhibitors and substrates possessing analogous dibasic consensus sequences have affinities for C subunit in the micromolar range(10, 11) . This discrepancy was partially explained in synthetic PKI peptide studies which extended the optimal inhibitory motif to include Phe, Arg, and Ile(6, 12) .

The molecular cloning of a cDNA from testis encoding a distinct isoform of PKI, PKI1, provided additional insight into the structural determinants of kinase inhibition(13, 14) . A cDNA encoding a second PKI isoform, PKI2, appears to represent an alternatively spliced mRNA which contains an amino-terminal extension of the PKI1 coding region(14) . While rat PKI1 shares only 41% amino acid identity with rabbit PKI(13) , the extended inhibitory motif of rabbit PKI is fully conserved in PKI1 and PKI2. However, despite the distinct developmental and tissue distributions of PKI and PKI1 mRNAs(15) , a rationale for the existence of multiple PKI isoforms has yet to be determined conclusively.

An earlier report demonstrated that rat PKI1 and rabbit PKI had similar K values(13) . However, murine PKI1 recently was shown to possess a 64-fold higher IC than human PKI(16) , providing evidence for a possible functional difference between PKI isoforms. To probe the structural cause of this difference in inhibitory potency, we produced recombinant PKI proteins that contained selected amino acid sequences from both murine PKI and PKI1. These chimeric PKIs were then subjected to kinetic analysis. Results from this study suggest that amino acid changes within the amino-terminal amphipathic -helix of murine PKI can fully account for the difference in inhibitory potency between the two murine PKI isoforms. Together, these experiments constitute the first mutational analysis of a PKI protein, and the first investigation of the structural basis for a difference in PKI isoform function.

MATERIALS AND METHODS

Construction of Chimeric PKI cDNAs and Site-directed Mutagenesis of Murine PKI1

To generate the PKI chimeras containing carboxyl-terminal swaps, cDNAs coding for the amino- and carboxyl-terminal residues of murine PKI and PKI1 were amplified in separate PCR reactions.

Reaction A

Reaction B

Reaction C

Reaction D

Reactions E and F yielded partially overlapping 5` and 3` fragments of murine PKI1 containing a complete -turn sequence from PKI. Altered nucleotides are underlined.

Reaction E

Reaction F

Reactions G and H produced partially overlapping 5` and 3` fragments coding for a murine PKI(1-12)/1(13-70) mutant.

Reaction G

Reaction H

Reactions I and J yielded partially overlapping 5` and 3` fragments coding for a murine PKI1(T8A/S12A) mutant. Altered nucleotides are underlined.

Reaction I

Reaction J

Reaction K yielded a full length fragment coding for a murine PKI1(I7Y) mutant. Altered nucleotides are underlined.

Reaction K

The reaction products (A, B), (C, D), (E, F), (G, H), and (I, J) were appended in reactions L-N.

Reaction L

Reaction M

Reaction N

The products of reactions K, L, M, and N were digested with EcoRI (Life Technologies, Inc.), and the resulting fragments were isolated and ligated into the EcoRI site of pMALcRI (18) (New England Biolabs). The pMALcRI plasmid expresses cDNA inserts as fusion proteins with maltose binding protein. The chimeric PKI and mutant PKI1 expression vectors were then electroporated into Escherichia coli XL1Blue (Stratagene). Individual clones were sequenced using a modified T7 DNA polymerase (Sequenase, U. S. Biochemical Corp.) in the dideoxy chain termination method (19) to verify mutations and to ensure that no additional modifications to the cDNAs were introduced.

The same steps described above were also used to amplify murine wild-type PKI using the cDNA template pGEM(PKI-7)Z and the oligonucleotides MSPKI.E5 and MSPKI.E3 and introduce it into the pMALcRI prokaryotic expression vector.

Expression and Purification of Maltose Binding Protein Fusion Proteins

Determination of PKI IC Values

Determination of PKI K Values

RESULTS AND DISCUSSION

Previous peptide studies revealed five amino acids in the amino terminus of PKI that are responsible for the majority of its potent inhibitory activity(6, 12) . The two pseudosubstrate site arginines, Arg and Arg, interact with residues within the active site of C subunit(5, 23) . The roles of the remaining critical amino acids, Phe, Arg, and Ile, became clear upon the elucidation of the crystal structure of C subunit complexed with synthetic PKI(5-24) peptide (23) . The aromatic side chain of Phe is positioned along an amphipathic -helix to interact with Tyr and Phe within a hydrophobic pocket in C subunit. Arg forms part of a -turn structure that positions its guanidinium group to ion-pair with Glu of C subunit. Ile is located within an extended portion of PKI(5-24) and interacts with a second hydrophobic pocket in C subunit.

Protein Sequence Comparison of Murine PKI and PKI1

Figure 1: Amino acid comparison of murine PKI and PKI1. The predicted amino acid sequences of murine PKI (top line) (17) and PKI1 (bottom line) (14) are shown. Boxed amino acids indicate identical residues between murine PKI and PKI1. Double arrows above the murine PKI sequence localize structural domains present in the PKI amino terminus as determined by x-ray crystallographic studies using a PKI(5-24) peptide(23) . The first four PKI amino acids (dotted line) were not included in the crystal structure, but may also participate in an -helix(25) .

Determination of K Values for Murine PKI and PKI1

Kinetic analyses of the murine PKI and PKI1 fusion proteins were performed using the method of Henderson for high affinity inhibitors (22) as described previously for PKI(6, 7, 12) . Henderson analysis demonstrates that murine PKI1, like all PKIs examined to date(13, 24) , is a competitive inhibitor of C (Fig. 2). Replots of slopes (Fig. 2, inset) from the Henderson analyses of both murine PKI and PKI1 versus Kemptide substrate (9) reveal a K for PKI1 of 7.1 nM, reflecting a 32-fold decrease in inhibitory potency compared to PKI (K = 0.22 nM). Since both murine isoforms possess the optimal PKI inhibitory motif, these results suggest that additional, nonconserved residues are responsible for the difference in K between PKI and PKI1.

Figure 2: Murine PKI1 and PKI K determinations by Henderson analyses. cAMP-dependent protein kinase activity was determined in the presence or absence of murine wild-type PKI1 or PKI at the following Kemptide concentrations: 90 µM (), 60 µM (), 30 µM (), and 5 µM (). Shown are the data for murine PKI1 plotted in accordance with Henderson (22) , where I is the total inhibitory protein concentration and V and V are the reaction velocities in the presence and absence of inhibitory proteins, respectively. The inset shows replots of the slopes from Henderson analyses versus Kemptide concentration for murine PKI1 () and PKI (). K values obtained from the replots are listed in Fig. 4.

Figure 4: Inhibition constants of wild-type and chimeric PKIs. The relative lengths of wild-type murine PKI (hatched bar) and PKI1 (solid bar) and the lengths and isoform compositions of the chimeric PKIs used in this study are shown on the left. All PKIs were expressed as fusion proteins with maltose binding protein (MBP), although cleavage of MBP from the fusion proteins did not affect the inhibitory activity of murine PKI and PKI1. Inhibition constants of the wild-type and chimeric PKIs are shown in the table to the right of the respective inhibitors. Inhibition constants are expressed as the average of three or more experiments ± S.D. for each PKI. K values for the chimeric PKIs were determined via Henderson analysis as described for wild-type murine PKI and PKI1.

Construction and Kinetic Analysis of PKI Chimeras

To assess the effect of the PKI mutagenesis on the inhibition of C, phosphotransferase activity was assayed in the presence of 30 µM Kemptide substrate and increasing concentrations of the chimeric PKIs (Fig. 3, A and B). Swapping the carboxyl-terminal two-thirds of murine PKI and PKI1 causes only minor changes in inhibitory activity (Fig. 3A). PKI(1-21)/1(22-70) demonstrates a respective 1.3- and 1.7-fold increase in IC and K relative to wild-type murine PKI (Fig. 4). PKI1(1-21)/(22-75) exhibits a respective 1.7- and 1.4-fold decrease in IC and K compared to wild-type murine PKI1 (Fig. 4). These modest effects suggest that nonconserved amino-terminal residues are responsible for the majority of the isoform-specific difference in inhibitory potency between murine PKI and PKI1.

Figure 3: Inhibition of C subunit by chimeric PKIs. C activity was assayed in the presence of 30 µM Kemptide and increasing concentrations of murine wild-type PKI (), PKI(1-21)/1(22-70) (), PKI1 (), and PKI1(1-21)/(22-75) () (A) or murine wild-type PKI (), PKI(1-12)/1(13-70) (), PKI1 (), and PKI1(A14G/A16T) () (B). Activity was determined as described under ``Materials and Methods'' and expressed as the percentage of C specific activity in the absence of inhibitor. The curves were fitted using the average values of triplicate assay points from representative experiments, and the error bars depict the standard deviation from the mean. The experiments were performed at least three times for each inhibitor. Average IC values and measurements of error for each PKI are reported in Fig. 4.

Since the pseudosubstrate site is conserved between the murine PKI isoforms, there remained only nine amino acid differences in the amino termini to investigate. Two of these residues in PKI, Gly and Thr, are located within a -turn. Gly, in particular, is thought to be important for turn formation or for providing flexibility for binding of Arg(23) . The importance of Arg interactions in full length PKI inhibition was investigated previously by mutating it to an alanine, which resulted in a large decrease in inhibitory activity(16, 20) . The remaining seven nonconserved residues in the amino terminus participate in an amphipathic -helix in PKI. The high affinity binding of PKI peptides is largely due to hydrophobic interactions between C subunit and residues along this helix, most notably Phe(16, 23) . Together, the -turn and -helix regions serve to fix the amino terminus of PKI, optimizing pseudosubstrate site contacts with active site residues in C subunit. To test whether either of these two PKI structural domains can account for the observed difference in K between the murine PKI isoforms, two additional PKI chimeras were examined.

PKI1(A14G/A16T), which contains the exact amino acid sequence corresponding to the -turn of PKI, does not demonstrate enhanced inhibitory activity relative to wild-type murine PKI1 (Fig. 3B). In fact, this chimeric PKI has IC and K values 1.5- and 1.1-fold greater than wild-type murine PKI1 (Fig. 4).

The final PKI chimera, PKI(1-12)/1(13-70), replaces the amino-terminal 12 amino acids of PKI1 with the exact amino acid sequence corresponding to the -helical region of PKI. Surprisingly, this chimeric PKI exhibits inhibition constants identical with wild-type murine PKI (Fig. 3B and 4).

The results of the chimeric PKI experiments suggest that an amino-terminal 12 amino acid region can fully account for the difference in murine PKI isoform inhibitory activity. At least part of this region forms an amphipathic -helix in PKI(23) , but the corresponding secondary structure in PKI1 is unknown. Nonconserved residues within the first 12 amino acids of murine PKI1 could decrease inhibitory activity through at least three different mechanisms. First, one or more of these residues may preclude the formation of an amphipathic -helix in PKI1. Second, murine PKI1 may possess an amino-terminal -helix whose amphipathicity differs from PKI. Third, murine PKI1 could possess an amphipathic -helix similar to PKI; however, variations in individual amino acid side chains may result in altered interactions with C subunit residues. The first mechanism is less probable, since the complete absence of an -helix would be predicted to severely disrupt interactions between Phe and its hydrophobic pocket in C subunit. The resulting decrease in inhibitory potency would likely be more striking than the 32-fold difference observed between murine PKI and PKI1, since direct mutation of Phe caused a 1200-fold increase in the IC of PKI(16) .

Inhibition of C Subunit by Amino-terminal Site-directed Mutants of Murine PKI1

Figure 5: Comparison of amino-terminal amino acid sequences of murine PKI and PKI1. A, comparison of the amino-terminal 12 amino acids of murine PKI and PKI1. Residues in bold were further investigated for their role in determining isoform-specific inhibitory activity. B, helical wheel diagram of the amino terminus of PKI. Residues in parentheses correspond to amino acid differences in murine PKI1. Specific amino acid positions examined in this study are designated with a filled circle.

The PKI mutant PKI1(T8A/S12A) replaces Thr and Ser in murine PKI1 with the alanine residues present in PKI. C phosphotransferase inhibition assays reveal an IC of 3.0 ± 1.0 nM for PKI1(T8A/S12A), reflecting a 4.0-fold decrease relative to wild-type murine PKI1 (Fig. 6). The fact that these mutations only partially improve the inhibitory potency of murine PKI1 suggests that Thr and/or Ser do not dramatically inhibit -helix formation. Hence, other nonconserved amino-terminal residues would be predicted to account for the remaining difference in the inhibition constants of murine PKI and PKI1. A strong candidate is Tyr, which is replaced by an isoleucine in murine PKI1. In a previous report utilizing synthetic PKI peptides, substitution at Tyr caused a greater increase in K than substitution of other nonconserved amino-terminal amino acids(12) . In addition, chemical modification of Tyr resulted in large decreases in the inhibitory potency of PKI(26) . Since Tyr is located adjacent to Phe in the amino-terminal -helix of PKI (Fig. 5B), it was speculated to interact with the hydrophobic pocket that accommodates Phe(23) .

Figure 6: Inhibition of C by murine PKI1 amino-terminal mutants. Recombinant C was assayed for phosphotransferase activity in the presence of 30 µM Kemptide and increasing concentrations of murine wild-type PKI (), PKI1 (), PKI1(T8A/S12A) (), and PKI1(I7Y) (). Activity was determined as described under ``Materials and Methods'' and expressed as the percentage of C specific activity in the absence of inhibitor. The curves were fitted using the average values of triplicate assay points from representative experiments, and the error bars depict the standard deviation from the mean. The experiments were performed at least three times for each inhibitor. Average IC values ± S.D. for the PKI mutants are discussed in the text.

In this study, site-directed mutagenesis of Ile of murine PKI1 to tyrosine yields an IC of 0.95 ± 0.45 nM, which constitutes a 13-fold increase in inhibitory activity (Fig. 6). Since isoleucines more strongly facilitate -helical structure than tyrosines(25) , PKI1(I7Y) is less likely to form an amino-terminal -helix than wild-type PKI1. Therefore, it would seem that specific interactions between the phenolic group of Tyr and C subunit residues are responsible for the enhanced inhibitory potency of murine PKI. This tyrosine residue was implicated as a potential PKI regulatory site in studies of in vitro phosphorylation by the epidermal growth factor receptor (27) . In these studies, phosphorylation at Tyr increased the IC of PKI by approximately 9-fold relative to dephosphorylated PKI, suggesting that interactions between Tyr and C subunit are important in C subunitPKI complex formation.

Results presented here imply a mechanism whereby C subunit can discriminate between various PKI isoforms. Specifically, we have shown that differences in amino acids such as Tyr, located in an amino-terminal region corresponding to the -helix of PKI, can result in distinct PKI inhibitory properties. Different C subunitPKI interactions may in turn lead to physiological changes in PKI function. Although a specific role for PKI has not been unequivocally assigned, recent studies by Fantozzi et al.(28) demonstrated that PKI can enhance C subunit export from the nucleus. Additional reports showed that full length PKI or a PKI peptide could preferentially inhibit basal transcriptional activity of some cAMP-regulated DNA response elements(29, 30) . Therefore, in an unstimulated cell with resting levels of cAMP, the difference in K between murine PKI and PKI1 could play a significant role in determining either the amount of C subunit present in the nucleus or the rate at which C subunit activates specific transcription factors. In particular, differential expression of PKI isoforms during development offers an intriguing mechanism to regulate the basal level of C subunit phosphotransferase activity(15) . While the exact number of PKI isoforms is not known, numerous chromatographically separable forms of PKI have been observed in testis (13, 31) . Of interest, one recently cloned murine PKI1 isoform, PKI2, possesses a unique amino terminus due to alternative splicing (14) . Although the inhibitory potency of PKI2 has not yet been examined, work here demonstrates that the existence of distinct amino-terminal amino acid sequences in multiple PKI isoforms may provide for further diversity in the regulation of cAMP-dependent protein kinase function.

FOOTNOTES

*

This work was supported by National Institutes of Health Grant GM 38788 (to M. D. U.). 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 and reprint requests should be addressed: Neuroscience Laboratories Bldg., University of Michigan, 1103 E. Huron St., Ann Arbor, MI 48104-1687. Tel.: 313-747-3172; Fax: 313-936-2690.

()

The abbreviations used are: PKI, cAMP-dependent protein kinase inhibitor; bp, base pair(s); PCR, polymerase chain reaction; C, catalytic; MBP, maltose binding protein.

ACKNOWLEDGEMENTS

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