Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4

The Ca(2+)-dependent protein kinases (CDPKs) are members of a large subfamily of protein kinases in plants that have been implicated in the control of numerous aspects of plant growth and development. One known substrate of the CDPKs is the ER-located ACA2 calcium pump, which is regulated by phosphorylation of Ser(45). In the present study, a synthetic peptide based on the known regulatory phosphorylation site (RRFRFTANLS(45)KRYEA) was efficiently phosphorylated in vitro by CDPKs but not a plant SNF1-related protein kinase. Phosphorylation of the Ser(45)-ACA2 peptide was surprising because the sequence lacks basic residues at P-3/P-4 (relative to the phosphorylated Ser at position P) that are considered to be essential recognition elements for CDPKs. We demonstrate that phosphorylation of the Ser(45)-ACA2 peptide is dependent on the cluster of basic residues found N-terminal (P-6 to P-9) as well as C-terminal (P + 1/P + 2) to the phosphorylated Ser. The results establish a new general phosphorylation motif for CDPKs: [Basic-Basic-X-Basic]-phi-X(4)-S/T-X-Basic (where phi is a hydrophobic residue). The motif predicts a number of new phosphorylation sites in plant proteins. Evidence is presented that the novel motif may explain the phosphorylation by CDPKs of Ser271 in the aquaporin PM28A.     

Nicotiana tabacum osmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop

NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress. Our previous results as well as data presented by others indicate that phosphorylation is involved in activation of SnRK2 kinases. Here, we have mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry with collision-induced peptide fragmentation. We show that active NtOSAK, isolated from NaCl-treated tobacco BY-2 cells, is phosphorylated on Ser-154 and Ser-158 in the kinase activation loop. Prediction of the NtOSAK three-dimensional structure indicates that phosphorylation of Ser-154 and Ser-158 triggers changes in enzyme conformation resulting in its activation. The involvement of Ser-154 and Ser-158 phosphorylation in regulation of NtOSAK activity was confirmed by site-directed mutagenesis of NtOSAK expressed in bacteria and in maize protoplasts. Our data reveal that phosphorylation of Ser-158 is essential for NtOSAK activation, whereas phosphorylation of Ser-154 most probably facilitates Ser-158 phosphorylation. The time course of NtOSAK phosphorylation on Ser-154 and Ser-158 in BY-2 cells subjected to osmotic stress correlates with NtOSAK activity, indicating that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. Importantly, Ser-154 and Ser-158 are conserved in all SnRK2 subfamily members, suggesting that phosphorylation at these sites may be a general mechanism for SnRK2 activation.     

Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.

Experiments were performed to investigated the mechanism of sucrose-phosphate synthase (SPS) activation by osmotic stress in darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immunopurification and was not the result of an increased SPS protein level. The previously described Ca(2+)-independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant enzyme catalyzing phosphorylation and inactivation of dephosphoserine-158-SPS. A new, Ca(2+)-dependent SPS-protein kinase activity (peak IV kinase) was also resolved and shown to phosphorylate and activate phosphoserine-158-SPS in vitro. The peak IV kinase also phosphorylated a synthetic peptide (SP29) based on the amino acid sequence surrounding serine-424, which also contains the motif described for the serine-158 regulatory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak IV kinase had a native molecular weight of approximately 150,000 as shown by gel filtration. The SP29 peptide was not phosphorylated by the inactivating peak III kinase. Osmotically stressed leaves showed increased peak IV kinase activity with the SP29 peptide as a substrate. Tryptic 32P-phosphopeptide analysis of SPS from excised spinach leaves fed [32P]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefore, at least part of the osmotic stress activation of SPS in dark leaves results from phosphorylation of serine-424 catalyzed by a Ca(2+)-dependent, 150-kD protein kinase.     

Phosphorylase kinase specificity. A comparative study with cAMP-dependent protein kinase on synthetic peptides and peptide analogs of glycogen synthase and phosphorylase

A synthetic pentadecapeptide, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-Leu-Pro-Gly-Leu-Glu, corresponding to the phosphorylatable site at the NH2 terminus of glycogen synthase, could be phosphorylated stoichiometrically at seryl residue 7 by both phosphorylase kinase and cAMP-dependent protein kinase. Phosphorylation of seryl residue 3 also occurred after prolonged incubation with cAMP-dependent protein kinase. Kinetic studies show that the pentadecapeptide is a better substrate for phosphorylase kinase. A peptide consisting of residues 1-11 was not as good a substrate and substitution of Arg-4 by Lys and Ser-9 by ARg in the unidecapeptide decreased and increased phosphorylase kinase reaction rates, respectively. Higher rates of phosphorylation were obtained with peptides of the phosphorylatable site of phosphorylase. A peptide with the sequence, Leu-Ser-Tyr-Arg-Arg-Tyr-Ser-Leu was phosphorylated initially by phosphorylase kinase and cAMP-dependent protein kinase at Ser-2 and Ser-7, respectively. Upon longer incubation, second site phosphorylation occurred with both kinases. A peptide of the same sequence with D-amino acids could not be phosphorylated but was a competitive inhibitor of both enzymes. The results suggest that optimal interaction of the two kinases depends on various factors including the orientation of arginyl groups with respect to the phosphorylatable serine.     

Substrate specificity of a multifunctional calmodulin-dependent protein kinase

The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide analogs. The enzyme phosphorylated a synthetic peptide corresponding to the NH2-terminal 10 residues of glycogen synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylated with a Vmax of 12.5 mumol X min-1 X mg-1 and an apparent Km of 7.5 microM compared to values of 1.2 mumol X min-1 X mg-1 and 3.1 microM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a Km of 4 microM and a Vmax of 5.4 mumol X min-1 X mg-1. The importance of the arginine 3 residues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased Vmax 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substrates and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other "arginine-requiring" protein kinases.     

Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate

1-Amino-cyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-determining step in the biosynthesis of the plant hormone ethylene, and there is evidence for regulation of stability of the protein by reversible protein phosphorylation. The site of phosphorylation of the tomato enzyme, LeACS2, was recently reported to be Ser460, but the requisite protein kinase has not been identified. In the present study, a synthetic peptide based on the known regulatory phosphorylation site (KKNNLRLS460FSKRMY) in LeACS2 was found to be readily phosphorylated in vitro by several calcium-dependent protein kinases (CDPKs), but not a plant SNF1-related protein kinase or the kinase domain of the receptor-like kinase, BRI1, involved in brassinosteroid signaling. Studies with variants of the LeACS2-Ser460 peptide establish a fundamentally new phosphorylation motif that is broadly targeted by CDPKs: phi -1-[ST]0- phi +1-X-Basic+3-Basic+4, where phi is a hydrophobic residue. Database analysis using the new motif predicts a number of novel phosphorylation sites in plant proteins. Finally, we also demonstrate that CDPKs and SnRK1s do not recognize motifs presented in the reverse order, indicating that side chain interactions alone are not sufficient for substrate recognition.     

Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro.

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.     

The influence of basic residues on the substrate specificity of protein kinase C

The substrate specificity of protein kinase C has been examined using a series of synthetic peptide analogs of glycogen synthase, ribosomal protein S6, and the epidermal growth factor receptor. The glycogen synthase analog peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala10 was phosphorylated at Ser7 with a Km of 40.3 microM. Peptide phosphorylation was strongly dependent on Arg4. When lysine was substituted for Arg4 the Km was increased approximately 20-fold. Addition of basic residues on either the NH2-terminal or COOH-terminal side of the phosphorylation site of the glycogen synthase peptide improved the kinetics of peptide phosphorylation. The analog Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was phosphorylated with a Km of 4.1 microM. Substitution of Ser7 with threonine increased the apparent Km to 151 microM. The truncated peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val8 was phosphorylated with similar kinetic constants to the parent peptide, however, deletion of Val8 increased the apparent Km to 761 microM. The ribosomal peptide S6-(229-239) was phosphorylated with a Km of approximately 0.5 microM predominantly on Ser236 and is one of the most potent synthetic peptide substrates reported for a protein kinase. The apparent Km for S6 peptide phosphorylation was increased by either deletion of the NH2-terminal 3 residues Ala229-Arg-231 or by substitution of Arg238 on the COOH-terminal side of the phosphorylation site with alanine. This analog peptide, [Ala238]S6-(229-239) was phosphorylated with an approximate 6-fold reduction in Vmax and a switch in the preferred site of phosphorylation from Ser236 to Ser235. These results support the concept that basic residues on both sides of the phosphorylation site can have an important influence on the kinetics of phosphorylation and site specificity of protein kinase C.     

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.     

Phosphorylation of bovine adrenodoxin. Structural study and enzymatic activity

Adrenodoxin is an iron-sulfur protein which functions as a carrier of reducing equivalents in steroid hydroxylation reactions catalyzed by specific cytochromes P-450 in steroidogenic tissues such as adrenal cortex. Purified bovine adrenocortical adrenodoxin was shown to be selectively phosphorylated upon incubation with purified cAMP-dependent protein kinase, whereas other protein kinases were ineffective. The phosphorylation reaction was completed within 45 min at 30 degrees C and resulted in the optimal incorporation of 1 mol phosphate/mol adrenodoxin. Apoadrenodoxin, lacking the iron-sulfur cluster, was also phosphorylated under similar conditions. An apparent Km of 55 microM with a Vmax of 0.3 pmol 32P incorporated min-1 mg adrenodoxin-1 was calculated. Phosphorylation resulted in a striking change in several molecular properties of adrenodoxin, such as electrophoretic behavior and hydroxyapatite affinity, thus providing the possibility of clearly separating phosphorylated from unphosphorylated adrenodoxin. In addition, phosphoadrenodoxin became refractory to mild trypsin degradation, whereas this was not the case with apoadrenodoxin. The phosphorylated site of adrenodoxin was identified as a serine residue; study of peptide products resulting from CNBr and proteolytic cleavages of phosphoadrenodoxin suggested that Ser-88 was the target of the phosphorylation reaction. The influence of phosphorylation upon adrenodoxin activity was examined using cholesterol side-chain cleavage and 11 beta-hydroxylase (11 beta) systems, reconstituted from purified components. Phosphorylation of adrenodoxin resulted in an average twofold decrease in its Km values for the two specific cytochromes P-450 involved. This effect was paralleled by a positive relationship between the degree of adrenodoxin phosphorylation and its ability to support the overall activity of reconstituted side-chain cleavage and 11 beta-hydroxylase systems. Although it remains to be examined whether adrenodoxin is phosphorylated in the intact cell, the present observations suggest that it represents a potential target in the hormonal regulation of the adrenocortical differentiated functions, especially by stimulatory agents acting through a cyclic-AMP-dependent mechanism, such as adrenocorticotropin.     

Troponin from the myocardium and skeletal muscles: structure and properties

The literary and experimental data on the structure and properties of cardiac and skeletal muscle troponin are reviewed. The cation--binding sites of cardiac and skeletal muscle troponin C are distinguished by specificity; the sites localized in the C-terminal part of the protein molecule can bind both Ca2+ and Mg2+, whereas the sites localized at the N-end specifically bind Ca2+. The use of bifunctional reagents revealed a number of helical sites within the structure of cardiac troponin C (residues 84-92 and 150-158) and of skeletal muscle troponin C (residues 90-98 and 125-136). A comparison of experimental data with the results of an X-ray analysis testifies to the presence in the central part of the troponin C molecule of a long alpha-helical sequence responsible for troponin C interaction with the inhibiting peptide of troponin I. The efficiency of interaction of troponin components depends on Ca2+ concentration; the integrity of the overall troponin complex is mainly provided for by troponin C interaction with troponin I and by troponin I interaction with troponin T. The interaction between troponins T and C is relatively weak, especially in the case of cardiac troponin components. Both skeletal and cardiac muscles synthesize several troponin T isoforms differing in length and amino acid composition of N-terminal 40-60 member peptides. Troponin T isoforms can undergo phosphorylation by several protein kinases. The single site of troponin T which exists in a phosphorylated state in vivo (residue Ser-1) undergoes phosphorylation by specific protein kinase (troponin T kinase) related to casein kinases II. It was assumed that the phosphorylation of Ser-1 residue of troponin T as well as the synthesis of troponin T isoforms differing in the structure of the N-terminal peptide, provides for the regulation of interaction between two neighbouring tropomyosin molecules.     

A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families

Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-theta and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.     

The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.     

Further studies on the phosphorylation and kinetics of rat liver fructose-1,6-bisphosphatase: importance of the three-dimensional structure of a substrate to protein kinase A.

Rat liver fructose-1,6-bisphosphatase was phosphorylated by cAMP-dependent protein kinase to 2.6 mol phosphate/mol subunit but not by Ca2+/phospholipid-dependent and Ca2+/calmodulin-dependent protein kinases. It was demonstrated that phosphorylation of Ser-341 and Ser-356, and to a much lower extent, Ser-338, was dependent on the presence of intact arginine residues. This observation implicates that the intact three-dimensional structure of the substrate is necessary for phosphorylation of Ser-356 since the closest arginine is located at a six amino acid residue distance.     

Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.     

Deciphering the Arginine-binding preferences at the substrate-binding groove of Ser/Thr kinases by computational surface mapping

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P-2 and P-5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P-2/P-5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P-5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P-2 and P-5 positions.     

Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C

Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.