©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase C-specific Phosphorylation of the Elongation Factor eEF-1 and an eEF-1 Peptide at Threonine 431 (*)

(Received for publication, September 22, 1994; and in revised form, December 7, 1994)

Kirsten Kielbassa Hans-Joachim Müller Helmut E. Meyer (1) Friedrich Marks Michael Gschwendt (§)

From the German Cancer Research Center, D-69120 Heidelberg, Federal Republic of Germany University of Bochum, D-44780 Bochum, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes alpha, beta, (cPKC) and PKC. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1alpha, is phosphorylated with much greater efficacy by PKC than by cPKC. Using the recombinant PKC isoenzymes alpha, beta, , , , , and , we could show that purified eEF-1alpha is indeed a specific substrate of PKC. It is not significantly phosphorylated by PKC, -, and - and only slightly by PKCalpha, -beta, and -. PKC phosphorylates eEF-1alpha at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence corresponding to that of 422-443 from murine eEF-1alpha and containing Thr-431 is an absolutely specific substrate for the -type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate by PKC and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete loss of selectivity.


INTRODUCTION

The PKC (^1)family, a group of 11 isoenzymes known so far possessing phospholipid-dependent serine/threonine kinase activity, plays a key role in cellular signal transduction and is involved in the regulation of numerous cellular processes and probably in tumor promotion (for reviews, see (1, 2, 3, 4) ). The PKC members can be subdivided into three groups. The conventional PKCs (cPKCs; alpha, beta(1), beta(2), and ) are Ca-responsive and activated by diacylglycerol or tumor-promoting phorbol esters such as TPA(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) ; the novel PKCs (nPKCs; , , , and ) are also activated by diacylglycerol or TPA but are Ca-unresponsive(1, 2, 3, 4, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) ; the atypical PKCs (aPKCs; , , and ) are Ca and diacylglycerol- and TPA-unresponsive(1, 2, 3, 4, 12, 26, 27, 28, 29, 30, 31, 32, 33) .

PKC isoenzymes are differentially expressed and respond differently to physiological stimuli in various tissues and cell types (for review, see (34) ). Therefore, it is likely that they serve different physiological purposes. Indeed, overexpression of various PKCs in different cell lines indicated that certain isoenzymes might induce a specific effect such as increased or reduced cell proliferation(35, 36, 37, 38, 39, 40) . Other experimental devices, such as overexpression of kinase-negative mutants, gene knock-out, or a permeabilization system to reconstitute PKC responses also provided some insight into cellular effects specifically induced by PKC isoenzymes(34) . However, the distinct functions of different isoenzymes in signaling processes, especially with respect to selective phosphorylation of substrates, are unknown to date. Some differences in the phosphorylation of a few commonly used PKC substrates by purified or partially purified PKC isoenzymes in vitro were observed previously (16, 17, 19, 20, 24, 26, 31, 32, 41). Furthermore, a few physiological substrates (42, 43, 44, 45, 46) were shown to be phosphorylated differentially by various PKC isoenzymes. Selective phosphorylation of a substrate by a single PKC isoenzyme has not been reported as yet.

Here we show that the elongation factor eEF-1alpha as well as an eEF-1alpha peptide are phosphorylated selectively by the -type of PKC isoenzymes and that the amino acid environment of the phosphorylation site, i.e. Thr-431, determines this selectivity.


EXPERIMENTAL PROCEDURES

Materials

TPA was kindly supplied by Prof. Dr. E. Hecker, German Cancer Research Center, Heidelberg, Germany. Recombinant baculoviruses containing sequences coding for the different PKC isoenzymes were a generous gift of Dr. S. Stabel, Max-Delbrück-Laboratorium, Köln, Germany. EF-1alpha from reticulocytes and an anti-EF-1alpha antiserum were provided by Dr. W. C. Merrick, Case Western Reserve University, Cleveland, OH. Various peptides were synthesized by R. Pipkorn, German Cancer Research Center, Heidelberg. Histone III-S, phosphatidylserine, and myelin basic protein were from Sigma. [-P]ATP (specific activity, 5000 Ci/mmol) was from Hartmann Analytics, Braunschweig, Germany. Q-Sepharose Fast Flow and S-Sepharose Fast Flow were from Pharmacia-LKB (Freiburg, Germany).

Methods

Phosphorylation Assay

Phosphorylation reactions were carried out in a total volume of 100 µl containing 20 mM Tris-HCl, pH 7.5, 50 mM beta-mercaptoethanol, 4 mM MgCl(2), 10 µg of phosphatidylserine, 10M TPA, various substrates as indicated in the text, 8 milliunits of purified PKC (PKC or cPKC) or extracts of baculovirus-infected insect cells expressing PKCalpha, -beta, -, -, -, -, and - (amount as indicated in the text), and a mixture of 18.75 µM ATP and 7.5 nM [-P]ATP (3.75 µCi). The assay mixture was incubated for 7 min at 30 °C, and the reaction was terminated by the addition of 250 µl of 10% trichloroacetic acid. After 30 min at 4 °C, the precipitated proteins were pelleted, redissolved in sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and visualized by autoradiography of the gels. For the quantification of phosphate incorporation, gel slides containing the phosphorylated protein were analyzed by liquid scintillation counting. To determine the phosphorylation of peptide substrates, the reaction was started by the addition of a modified ATP mixture of 37.5 µM ATP and 1.5 nM [-P]ATP (0.75 µCi). After incubation for 7 min at 30 °C, the reaction was terminated by transferring 50 µl of the assay mixture onto a 20-mm square piece of phosphocellulose paper (Whatman p81), which was washed 3 times in deionized water and twice with acetone. The radioactivity on each paper was determined by liquid scintillation counting.

Purification of PKC and cPKC was performed as described elsewhere (15, 47) .

Recombinant PKC Isoenzymes

Sf9 cells were infected with recombinant baculoviruses and the cell extracts were used as a source for the isoenzymes as described previously(48) .

Purification of eEF-1alpha

All operations were carried out at 0-4 °C unless otherwise indicated. 20 g of porcine spleen was homogenized in 290 ml of buffer A (1 mM EDTA, 50 mM beta-mercaptoethanol, 50 mM Tris-HCl, pH 8.0) using a Waring blender (3 times 15 s). The homogenate was centrifuged at 100,000 times g for 30 min, and the resulting supernatant (cytosol) was applied to a Q-Sepharose Fast Flow column (2.6 times 26 cm), equilibrated with buffer A. The column was washed with 2 volumes of buffer A with a flow rate of 5 ml/min. The absorbance at 280 nm was monitored. Fractions of 10 ml were collected, and 30-µl aliquots were phosphorylated by PKC and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The void volume fractions with the highest content of EF-1alpha were pooled and applied to a S-Sepharose Fast Flow column (1.6 times 10 cm) equilibrated with buffer A. The column was washed with 1 volume of buffer A plus 100 mM NaCl, and protein was eluted with a gradient from 10 to 100% of 1 M NaCl in buffer A with an increase of 0.82%/min. The flow rate was 2 ml/min, and fractions of 2 ml were collected. eEF-1alpha eluted at 180 mM NaCl. Fractions containing exclusively eEF-1alpha were pooled and concentrated to 1.5 ml using a Centriprep 10 (Amicon).

Partial Purification of Annexin I

Purification was performed as described for eEF-1alpha. The void volume of the S-Sepharose Fast Flow column contained partially purified annexin I.

Phosphoamino Acid Analysis

Phosphorylated eEF-1alpha was precipitated with 10% trichloroacetic acid and hydrolyzed in 6 N HCl at 110 °C for 2 h. The sample was evaporated to dryness, dissolved in 25 µl of water containing the marker phosphoamino acids, and electrophoresed on Whatman No. 3MM paper in acetic acid/pyridine/water (52:5:943) for 2.5 h (250-450 V).

Determination of Phosphorylation Site(s) in eEF-1alpha

Phosphorylation and Tryptic Digestion of eEF-1alpha

An assay mixture of 1 ml contained 2.6 nmol of eEF-1alpha, 0.083 nmol of PKC (sufficient to incorporate 370 nmol of phosphate/min into histone III-S under similar conditions), 92.5 µg of phosphatidylserine, 20 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), 10M TPA, and 17.9 nmol [-P]ATP (71.5 µCi). The mixture was incubated for 60 min at 30 °C, and the reaction was terminated by the addition of 70 µl of trichloroacetic acid. The pellet was washed twice with the same volume of ice-cold acetone, and the dried protein pellet was dissolved in 15 µl of 6 M guanidinium-HCl, 2 mM dithiothreitol, 20 mM Tris-HCl, pH 8.0. Two aliquots of 3 µl each were removed for determination of protein content and radioactivity. The remaining sample was diluted with 111 µl of 1 mM CaCl(2), 50 mM Tris-HCl, pH 7.6 (to reduce the guanidinium concentration to 0.7 M), and subjected to digestion (35.7 °C, 24 h) with trypsin at the ratio 1:5.

Purification and Sequence Analysis of Tryptic Phosphopeptides

The digest was injected directly into an analytical (4.6 times 250 mm) SGE (Scientific Glas Engineering, D-Weiterstadt) HPLC column equilibrated with 0.1% trifluoroacetic acid/H(2)O. The gradient was run from 0 to 59% acetonitrile at an increase rate of 1%/min with a flow rate of 0.4 ml/min. The absorbance was monitored at 215 and 295 nm, and peak fractions were collected. P-labeled phosphopeptides were detected by liquid scintillation counting and diluted with two volumes of H(2)O to reduce the acetonitrile concentration. Phosphopeptide fractions were further purified by a second chromatographic step on an analytical C(18) RP column (2 times 250 mm), equilibrated with 10 mM ammonium acetate, pH 6.0. Peptides were eluted by a linear gradient from 0 to 59% acetonitrile with a flow rate of 80 µl/min.

The amino acid sequence of phosphopeptides was determined by automated Edman degradation in an Applied Biosystems, Inc. (Foster City, CA) model 473A protein sequencer. Phosphothreonine was identified as described by Meyer et al.(49) .

SDS-polyacrylamide gel electrophoresis was performed on 9% gels, and protein content was determined as described previously(50) .


RESULTS

PKC Isoenzyme-specific Protein Phosphorylation in Tissue Extracts

It was our objective to search for PKC substrates that might be phosphorylated preferentially by one of the PKC isoenzymes. In order to investigate substrate phosphorylation of tissue extracts (e.g. cytosol of murine epidermis or porcine spleen) with purified PKC in vitro, endogenous PKC had to be removed. This could be achieved by chromatography of the cytosol on Q-Sepharose, which bound PKC and other protein kinases. As shown in Fig. 1, proteins in the void volume of the Q-Sepharose column were not phosphorylated with [P]ATP in the absence of exogenous kinase. Only after addition of purified PKC (cPKC (alpha, beta, ) or PKC) phosphorylated proteins could be observed. In this and the following experiments with cPKC and PKC, the enzymes were used at equal activities based on the phosphorylation of histone III-S. Equalization of the enzyme activities with myelin basic protein or protamine sulfate gave essentially the same results. A 37-kDa protein (p37) was phosphorylated preferentially by cPKC, whereas PKC favored a 50-kDa protein (p50). After partial purification of p37 from the Q-Sepharose void volume by chromatography on S-Sepharose, it could be identified with annexin I by immunoblotting with an anti-annexin I antiserum (data not shown). Following chromatography on Q-Sepharose, p50 was purified almost to homogeneity by chromatography on S-Sepharose (Fig. 2) and could be identified with the elongation factor eEF-1alpha by comparison with authentic eEF-1alpha. Immunoblotting of p50 and eEF-1alpha with an anti-eEF-1alpha antiserum as well as autoradiography after phosphorylation of both proteins by PKC showed that p50 was identical with eEF-1alpha (Fig. 3). The weak intensities of the eEF-1alpha bands, as compared with those of p50, were due to the available low amount of authentic eEF-1alpha. P50 was found to be an extremely basic protein with a pI of 9 (data not shown). This pI is characteristic for eEF-1alpha and served as further evidence for the identity of p50 and eEF-1alpha. Moreover, the sequence of a tryptic peptide of p50 was found to be identical with the sequence 428-438 of eEF-1alpha (see below).


Figure 1: Chromatography of cytosol from porcine spleen on Q-Sepharose and differential phosphorylation of p37 and p50 in the void volume by cPKC and PKC. Chromatography and phosphorylation of proteins (40 µg) were performed as described under ``Methods.'' Phosphorylated proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.




Figure 2: Purification of p50 from the Q-Sepharose void volume by chromatography on S-Sepharose (see ``Methods''). Purified p50 in fractions 11, 12, and 13 of the S-Sepharose eluate (NaCl gradient; 30-µl aliquots) was phosphorylated with PKC run on an SDS-polyacrylamide gel and visualized by autoradiography and Coomassie Blue staining.




Figure 3: Identification of p50 as eEF-1alpha. Comparison of purified p50 with authentic eEF-1alpha by immunoblotting with an anti-eEF-1alpha antiserum and by autoradiography of the PKC-phosphorylated proteins. Alkaline phosphatase-conjugated goat anti-rabbit IgG was used for immunostaining. Immunoblot: 10 µg of p50, 0.4 µg of eEF-1alpha; Autoradiogram: 10 µg of p50, 0.2 µg of eEF-1alpha. A limited amount only of authentic eEF-1alpha was available. The intensity of eEF-1alpha in the immunoblot was found to be around 30% and in the autoradiogram around 8% of that of p50 and thus more than expected. This was probably due to a nonlinear relationship between the amount of protein and the intensity of either immune reaction or autoradiography.



The differential phosphorylation of eEF-1alpha by cPKC and PKC was studied in more detail. Fig. 4shows the time dependence of the eEF-1alpha phosphorylation by cPKC and PKC. After 60 min, when saturation was reached, eEF-1alpha had incorporated around 7 times more phosphate in the presence of PKC (0.4 mol of phosphate/mol of eEF-1alpha) than in the presence of cPKC. The K(m) and V(max) values of the phosphorylation by PKC were 0.21 µM and 4.21 nmol of phosphate/min/mg and of the phosphorylation by cPKC 0.27 µM and 0.37 nmol of phosphate/min/mg, respectively (Table 1). Thus, the concentration of eEF-1alpha in a tissue (about 20 µM) is 100-times greater than the K(m). The V(max)/K(m) value of the phosphorylation by PKC was about 15 times higher than that by cPKC. The finding that eEF-1alpha was selectively phosphorylated by PKC became even more evident using various recombinant PKC isoenzymes (alpha, beta, , , , , and ) from baculovirus-infected insect cells. The isoenzymes were used at equal activities based on phosphorylation of either protamine sulfate or myelin basic protein. In both cases, PKC was by far the most effective isoenzyme (Fig. 5). Other Ca-unresponsive isoenzymes (, , and ) did not phosphorylate eEF-1alpha, whereas the Ca-responsive isoenzymes (alpha, beta, and ), in accordance with the results obtained with cPKC, phosphorylated eEF-1alpha slightly. The extremely intense autophosphorylation of PKC (see Fig. 5) has previously been observed with the purified enzyme from porcine spleen, which incorporated around 4 times as much phosphate as cPKC (15) . (^2)


Figure 4: Time dependence of eEF-1alpha phosphorylation by cPKC and PKC. 2.5 µg of eEF-1alpha were phosphorylated with 8 milliunits of each of cPKC (circle) or PKC (bullet) for the times indicated as described under ``Methods.''






Figure 5: Phosphorylation of eEF-1alpha by various recombinant PKC isoenzymes. 2.5 µg of eEF-1alpha were phosphorylated with extracts of baculovirus-infected insect cells expressing PCKalpha, beta, , , , or , as described under ``Methods.'' Equal activities of the isoenzymes were used as determined by phosphorylation of myelin basic protein or protamine sulfate. Depending on the isoenzyme's activity, the extracts added to the assay contained 5-25 µg of protein. PKC was used only in one of the two experiments (upperpart of the figure).



Identification of a PKC-specific Phosphorylation Site in eEF-1alpha

The analysis of phosphoamino acids after hydrolysis of PKC-phosphorylated eEF-1alpha showed that phosphate was incorporated only into threonine (data not shown). After phosphorylation of eEF-1alpha with PKC and digestion with trypsin, P-labeled tryptic peptides were separated by HPLC (Fig. 6A), further purified by a second HPLC (Fig. 6B), and sequenced. As shown in Fig. 6A, two labeled peptides (A and B) corresponding to sequences 428-438 and 430-438 (based on the sequence of murine eEF-1alpha) were found, both containing Thr-431 as the phosphorylated amino acid. The peptide at about 15 min in the first HPLC run (Fig. 6A) was partially sequenzed and could be shown to be an impurity. The peptide just before B (Fig. 6, A and B) was found to be a peptide B with a modified methionine. 63% of total radioactivity was associated with the phosphorylation of Thr-431. Based on this as well as the phosphoamino acid analysis and the amount of phosphate incorporated (see above), Thr-431 was the only phosphorylated amino acid in PKC-treated eEF-1alpha. Taken together, these results indicate that Thr-431 with its amino acid environment provides a specific phosphorylation site for PKC.


Figure 6: Separation of tryptic eEF-1alpha peptides by HPLC and determination of amino acid sequences of phosphorylated peptides. PKC-phosphorylated eEF-1alpha was digested with trypsin; tryptic peptides were separated by two HPLC runs (first run, A; second run, B); and the amino acid sequence of the phosphorylated peptides A and B were analyzed as described under ``Methods.'' OD215 (-), acetonitrile (bulletbulletbulletbullet), cpm (-bullet-bullet-bullet).



Identification of a PKC-specific Peptide Substrate

In order to investigate this PKC-specific phosphorylation site in more detail, we synthesized the peptide RFAVRDMRQTVAVGVIKAVDKK (peptide 1) with a sequence corresponding to sequence 422-443 of murine eEF-1alpha containing Thr-431 and tested it as a substrate for purified cPKC and PKC as well as for various recombinant PKC isoenzymes (alpha, beta, , , , , and ). Peptide 1 was a suitable substrate for PKC (Fig. 7). As judged by the K(m) (22.9 µM), V(max) (118 nmol of phosphate/min/mg) and V(max)/K(m) (5.2) values, however, it was a less efficient substrate than eEF-1alpha (see Table 1). No significant phosphorylation was observed with cPKC. The recombinant isoenzymes beta, , , , and did not phosphorylate the peptide at all, and PCKalpha incorporated only a very small amount of phosphate into the peptide (around 10 times less than PKC (Fig. 8)). Contrary to the phosphorylation by PKC and similar to that by cPKC (see Fig. 8), the extent of phosphorylation of the peptide by PKCalpha was low over the entire substrate concentration range tested (data not shown). Thus K(m) and V(max) values could not be determined. Taken together, the peptide was even more selectively phosphorylated by PKC than protein eEF-1alpha (compare Fig. 5). In order to study the role of basic amino acids in the vicinity of Thr-431, we used peptides with various mutations in these basic amino acids. In peptide 2 (RRFAVRDMAQTVAVGVIKAVDKK) Arg-429 of peptide 1 was exchanged for alanine. The isoelectric point (pI, 11.7) of the peptide was kept constant by the addition of 1 arginine to the N terminus of the peptide. The lack of Arg-429 completely abolished the ability of PKC to phosphorylate the peptide (data not shown). Thus the basic amino acid in closest position to Thr-431 is essential for the recognition of the peptide as a substrate by PKC. A shift of Arg-429 to a position only 1 amino acid further away from Thr-431 than in peptide 1 (exchange of Arg-429 with Met-428) caused a significant decrease in the phosphorylation of this peptide (peptide 3) by PKC (Fig. 8). In peptide 4 (MFAVRDRRQTVAVGVIKAVDKK) the N-terminal Arg-422 of peptide 1 was exchanged with Met-428, and in peptide 5 (MFAVRDRRQTVKKGVIKAVDAV), additionally, the C-terminal Lys-442 and Lys-443 of peptide 1 were exchanged with Ala-433 and Val-434, respectively. Consequently, in peptide 4 and 5, Thr-431 was flanked closely by 2 basic amino acids either on one side or on both sides rather than by only 1 basic amino acid on one side in the original peptide 1. Again, the isoelectric point of the peptides was not changed by the mutation. Peptide 4 was phosphorylated most efficiently by PKC but to some extent also by PKCalpha, beta, and . Contrary to peptides 1 and 4, peptide 5 was phosphorylated very efficiently by all PKC isoenzymes (Fig. 8). The K(m) and V(max) values of the PKC phosphorylation of peptide 5 were 6.5 µM and 1103 nmol of phosphate/min/mg, respectively. Peptide 5 was 33 times more efficient as a substrate for PKC than peptide 1, as judged by the V(max)/K(m) values (see Table 1). Thus, this increase in the basic environment improved the efficacy of Thr-431 as phosphorylation site for all PKC isoenzymes and abolished its specificity for PKC.


Figure 7: Phosphorylation of the eEF-1alpha petide (petide 1) by 8 milliunits each of cPKC (circle) or PKC (bullet). For details of the procedure, see ``Methods.''




Figure 8: Phosphorylation of the eEF-1alpha peptide 1 and the mutated petides 3, 4, and 5 (5 nmol each) by recombinant PKC isoenzymes (see Fig. 5). Equal activities of the isoenzymes based on the phosphorylation of myelin basic protein were used. Peptide 1: RFAVRDMRQTVAVGVIKAVDKK; peptide 3: RFAVRDRMQTVAVGVIKAVDKK; peptide 4: MFAVRDRRQTVAVGVIKAVDKK; peptide 5: MFAVRDRRQTVKKGVIKAVDAV.




DISCUSSION

In the cell, the different PKC isoenzymes are thought to have a distinct function regarding phosphorylation of specific substrate proteins. Selective substrate phosphorylation could be due to an intrinsic specificity of an isoenzyme and/or to a specificity acquired by modification of an isoenzyme (e.g. by phosphorylation) and/or to a specific localization of an isoenzyme. Very recently, we have shown that PKC is phosphorylated in vitro by Src and that this tyrosine phosphorylation induces a stimulation of PKC activity apparently exhibiting some substrate selectivity(51) . Specific intracellular localizations of different PKC isoenzymes were reported, but a correlation with selective substrate phosphorylation has not been demonstrated as yet. Various purified or partially purified PKC isoenzymes were tested in vitro for substrate selectivity, and some differences in the phosphorylation of substrates were observed. Most frequently, however, only histone, myelin basic protein, protamine sulfate, and pseudosubstrate-related peptides were used as substrates (16, 17, 19, 20, 24, 26, 31, 32, 41). There are a few reports on other substrates, such as glycogen synthase kinase-3beta(42) , epidermal growth factor receptor(43) , vitamin D receptor(44) , neuromodulin(45) , and Raf(46) , which appear to be preferentially phosphorylated in vitro by one or the other PKC isoenzyme. However, selective phosphorylation of a substrate by a PKC isoenzyme has not been reported as yet.

We tried to find isoenzyme-specific substrate proteins in tissue extracts and were able to show that a 50-kDa protein in the cytosol of murine epidermis or porcine spleen is phosphorylated to a much greater extent by PKC than by cPKC (PKCalpha, -beta, and -). The significance of this observation is supported by the concomitant finding that another protein (p37, identified as annexin I) is phosphorylated much more efficiently by cPKC than by PKC. This indicates the existence of a differential phosphorylation of some proteins by different PKC isoenzymes. p50 could be identified as the elongation factor eEF-1alpha by comparison with authentic eEF-1alpha and by immunoblotting with an anti-eEF-1alpha antibody. Moreover, the sequence of a tryptic peptide of p50 is identical with the sequence 428-438 of eEF-1alpha (see below). According to our results obtained with various recombinant PKC isoenzymes from baculovirus-infected insect cells, purified eEF-1alpha is most effectively phosphorylated by PKC, only slightly by PKCalpha, -beta, and -, and not at all by PKC, -, and -. The amount of phosphate incorporated into eEF-1alpha by PKC indicates the existence of only one phosphorylation site, and, according to the phosphoamino acid analysis, this single site is a threonine. In accordance with these results, sequence analysis of purified tryptic peptides of phosphorylated eEF-1alpha revealed that Thr-431 is the sole amino acid in eEF-1alpha, which is phosphorylated by PKC. The physiological significance of this phosphorylation is not known as yet. Phosphorylation of eEF-1alpha by a partially purified PKC preparation (probably a mixture of PKCalpha, -beta, and -) in vitro and in intact cells upon treatment with TPA was reported previously(52, 53) . Phosphorylation in vitro of the subunit eEF-1alpha alone, i.e. not complexed with the other subunits of eEF-1alpha (beta and ) did not affect its translation activity. However, besides its role as elongation factor in protein biosynthesis, eEF-1alpha appears to exhibit still other functions (for review, see (54) ). It was shown to play a role in cell cycle-associated processes(55, 56) , in ubiquitin-regulated degradation of proteins(57) , in aging(58, 59) , and as a regulator of cytoskeletal rearrangements(56, 60, 61) . It is conceivable that some of these activities of eEF-1alpha are modified by its phosphorylation.

Knowing the phosphorylation site, we became interested in the question as to whether the amino acid environment of Thr-431 was responsible for the PKC-specific phosphorylation of Thr-431 and whether this isoenzyme specificity could be demonstrated in an appropriate synthetic peptide. Most intriguingly, a peptide corresponding to sequence 422-443 of murine eEF-1alpha and containing Thr-431 (peptide 1) is phosphorylated with high specificity by PKC, i.e. other isoenzymes do not accept this peptide as a substrate. It is conceivable that this peptide may be used to selectively determine PKC activity in crude tissue or cell extracts. This possibility is presently being investigated. It is well known that phosphorylation sites of PKC substrates require basic amino acids in the vicinity of serine or threonine that become phosphorylated. Consensus phosphorylation site motifs for PKC were defined, such as (K/R)XX(S/T), (K/R)X(S/T), (K/R)XX(S/T)X(K/R), etc.(62) . Turner et al.(63) reported on the differential phosphorylation of synthetic peptides derived from bovine myelin basic protein and some mutated forms of these peptides by a PKC preparation from pig brain (probably a mixture of PKCalpha, -beta, and -). Marais et al.(64) performed a systematic investigation of sequence requirements for synthetic peptides functioning as substrates for PKCalpha, -beta(1), and -. However, respective studies with isoenzymes of the nPKC group have not been reported, and a peptide with an amino acid sequence recognized selectively by a PKC isoenzyme has not been described as yet. The eEF-1alpha peptide with the PKC-specific phosphorylation site contains only 1 basic amino acid (Arg-429) in close vicinity to Thr-431. This is in accord with (K/R)X(S/T) being one of the consensus phosphorylation site motifs for PKC(62) . Upon removal of this basic amino acid, the peptide (peptide 2) is no longer able to serve as a substrate for PKC. Even a slight shift of this basic amino acid just one position away from the phosphorylation site (peptide 3) causes a significant decrease of the phosphorylation by PKC. This clearly demonstrates that the minimal requirement of PKC for the phosphorylation site in the eEF-1alpha peptide is the presence of a single basic amino acid close to the phosphate-accepting amino acid. Obviously, the minimal requirement of the other PKC isoenzymes for this phosphorylation site is different since they do not recognize the eEF-1alpha peptide as a substrate. We assume that phosphorylation sites of the other isoenzymes might require more than 1 basic amino acid. Indeed, a mutated eEF-1alpha peptide with 2 basic amino acids on either side of Thr-431 (428-429 and 433-434; peptide 5) is phosphorylated by all PKC isoenzymes. Moreover, the mutated peptide is phosphorylated by PKC much more efficiently than the original peptide 1. Surprisingly, a mutated eEF-1alpha peptide with 2 basic amino acids only on one side of Thr-431 (428-429; peptide 4) does not show the drastic increase in phosphorylation by all PKC isoenzymes that is observed with peptide 5. This indicates that PKC, like the other PKC isoenzymes, prefers phosphorylation sites with a strong basic environment but, at least with some substrates, tolerates weak basic sites better than the other isoforms. Probably due to this property, PKC is able to phosphorylate eEF-1alpha and possibly also other substrates selectively.


FOOTNOTES

*
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.: 49-6221-424505; Fax: 49-6221-424406.

(^1)
The abbreviations used are: PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; HPLC, high performance liquid chromatography.

(^2)
K. Kielbassa, F. Marks, and M. Gschwendt, unpublished results.


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