©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutation of Serine 90 to Glutamic Acid Mimics Phosphorylation of Bovine Prolactin (*)

(Received for publication, August 21, 1995)

Patricia M. Maciejewski Francis C. Peterson Patricia J. Anderson Charles L. Brooks (§)

From the Department of Veterinary Biosciences, the Ohio State Biochemistry Program, and the Department of Chemistry, The Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosphorylated prolactin has been identified and isolated from bovine pituitaries. The biological activity of this phosphoprotein is severely reduced in comparison with nonphosphorylated prolactin. The sites of phosphorylation are serines 26, 34, and 90, and the stoichiometry is 1:1:10, respectively. In this report, the phosphoserine residues have been individually replaced with glutamic acid in recombinant methionyl bovine prolactins in order to mimic phosphorylation at each site. Substitution of glutamic acid for serine at positions 26, 34, and 90 reduced protein helical contents by 10, 6, and 14%, respectively. UV absorbances for S26E and S34E bovine prolactins were blue-shifted, similar to the biological isolates of phosphorylated bovine prolactin, but the biological activities of the S26E and S34E mutants (ED values of 16.3 and 18.8 pM, respectively) were similar to that of wild-type prolactin (ED value of 18.6 pM) in the Nb2 rat lymphoma assay. S90E bovine prolactin had the greatest reduction in helical content but showed similar UV and fluorescent spectra to the wild-type bovine prolactin. The biological activity of S90E bovine prolactin (ED value of 672 pM) was reduced to an activity similar to that of phosphorylated bovine prolactin. The data indicate that the phosphorylation of serine 90 is responsible for the reduction in biological activity.


INTRODUCTION

Prolactin binds and activates receptors on the plasma membrane of target cells(1) . The PRL (^1)receptor is a member of the cytokine receptor superfamily(2) , receptors with extracellular and intracellular domains connected by a single transmembrane domain. Several receptors in this superfamily activate cells by formation of receptor dimers brought about by the binding of two receptors binding to unique surfaces of the ligand (sites 1 and 2)(3) . Receptor dimerization promotes the association and/or activation of the Janus family of tyrosine kinases (4) and phosphorylation of specific tyrosines of the receptor and substrate proteins that regulate transcription in the nucleus or the activity of other kinase systems. Substrate proteins appear to be presented to the Janus kinases by binding to the phosphorylated loci of the intracellular domain of the receptor(5) .

The three-dimensional structure of PRL has not been solved, but it is likely to be a four-helix bundle motif (6) similar to the up-up-down-down structure of hGH, a protein related by sequence able to bind prolactin receptors and induce lactogenic actions. The sequence of several variants of the PRL receptor have been reported; most variation between receptor subtypes is observed in the intracellular domain(7) . The three-dimensional structure of hGH bound to the extracellular domain of the hPRL receptor in a 1:1 complex has been reported(8) . These data have defined the site 1 surface area and have shown that the four-helix bundle of hGH has undergone little structural change when bound to either the hPRL or hGH receptor. However, there is a major shift of the first ancillary helix of the loop connecting helices 1 and 2. The surface areas of hGH that contact the extracellular domains of either hPRL or hGH receptors substantially overlap(9) . A limited number of residues within the contact areas provide the specificity for the lactogenic and somatotropic receptor binding by participating in specific interactions that provide most of the free energy liberated during the binding reaction(10) . Receptor binding site 2 for hPRL is proposed to be within the channel between helices 1 and 3(11) .

From this information, one can hypothesize that similar surfaces of PRLs and hGH constitute sites 1 and 2 that bind receptors to create the 1:2 hormone:receptor heterotrimeric complex. If this is the case, then the most likely surfaces for receptor binding determinants for PRLs are on helices 1, 3, and 4 and the long loop connecting helices 1 and 2. Several studies with PRL have identified functional determinants that are consistent with this idea(12, 13) .

Phosphorylated PRL has been identified in rat, turkey, and cow(6) . Phosphorylated PRLs appear to have altered structure, because they have unique biological activities or are unable to bind or activate PRL receptors to initiate biological actions(14, 15) . The addition of exogenous phosphorylated rat PRL to primary cultures of rat pituitary cells reduces secretion of endogenous PRL(16) . Phosphorylated rat (17) or bovine (15) PRL is not active in the Nb2 rat lymphoma bioassay(18) , whereas the nonphosphorylated form is mitogenic. Enzymatic removal of the phosphate will restore the potency of phosphorylated bPRL measured in this assay(15) . Phosphorylated bPRL does not compete with I-labeled nonphosphorylated bPRL for binding to the intermediate form of the PRL receptor found in Nb2 cells(15) . The failure of phosphorylated bPRL to bind to the PRL receptor suggests that phosphorylation removed the binding determinants of site 1 from the spatial relationship required for receptor binding(19) ; in other words, phosphorylation has induced a conformation change.

Phosphorylated bPRL has been isolated and characterized from bovine pituitaries(20, 21) . Three sites of phosphorylation were identified at serines 26, 34, and 90(20) . Phosphorylated bPRL has physical properties that are different from the nonphosphorylated form and are believed to be a consequence of phosphorylation. Stoichiometric studies indicate that serine 90 is phosphorylated approximately ten times more often than serines 26 or 34; thus, the unique physical and biological properties of phosphorylated bPRL are most likely due to phosphorylation at this residue. Serine 90 of bPRL is conserved in PRLs, GHs, and placental lactogens, whereas serines 26 and 34 are only found in PRLs. Serine 90 is distal from the documented and proposed binding determinants of lactogenic hormones(8, 13, 22) ; thus, if phosphorylation at this site is responsible for the loss of biological activity, then it must induce sufficient distal conformation change to remove the binding determinants from their spatial relationship required to successfully engage the PRL receptor.

Elucidation of the specific role of each phosphorylation site is not possible using the biological isolates of bPRL as they contain mixtures of several phosphorylated variants. Recently, several investigators have substituted serine with acidic residues in recombinant proteins to mimic the effects of phosphorylation. Substitution of glutamic or aspartic acid for serine 113, the site of phosphorylation in isocitrate dehydrogenase, blocks the binding of isocitrate in the active site of this protein and reduces enzymatic activity(23, 24) . Trautwein et al.(25) have substituted aspartic acid for serine 105 in the transcription activator NF-IL6/LAP and have mimicked the effect of phosphorylation in activation of a NF-IL6/LAP-regulated construct. In addition, alanine substitution of serine 105 eliminated the enhancement of transcription observed with the addition of exogenous kinase to an in vitro system.

We have substituted glutamic acid for serines 26, 34, and 90 to determine if mimicry of phosphoserine residues will produce changes in the properties and biological actions of bPRL that are similar to those produced by phosphorylation. By this approach we hope to elucidate the roles of individual phosphorylation sites.


MATERIALS AND METHODS

Plasmids and Bacterial Strains

Plasmid pT7-7 was kindly provided by S. Tabor (Harvard Medical School, Boston, MA). An f(1) origin of replication was ligated into the plasmid at the ClaI site to obtain both positive and negative strand vectors. (^2)The positive strand pT7-7 phagemid was used in all studies. E. coli strains DH5alpha, RZ1032 dutung, and BL21(DE3) were used for cloning, production of uridine-substituted single-stranded DNA for in vitro mutagenesis, and protein expression, respectively.

Preparation of Recombinant Bovine Prolactin

Total RNA was isolated from bovine anterior pituitary using RNAsol B (Tel-Test, Inc., Friendswood, TX). cDNA was prepared with reverse transcription with random hexamers. The 5` primer for polymerase chain reaction was designed to eliminate the leader sequence, add a methionyl residue at the start of the mature sequence, and provide an NdeI site at the methionyl residue. The first 7 residues were converted to bacterially preferred codons by design of this primer. The 3` primer introduced a HindIII restriction site following the termination codon. The polymerase chain reaction product was digested with NdeI and HindIII and ligated into the pT7-7 phagemid previously cut by the same restriction enzymes. The product was transformed into DH5alpha cells, and colonies were selected after growth on LB agar plates containing ampicillin. Selected colonies were initially analyzed by agarose gel electrophoresis. Positive colonies contained a 3623-base pair DNA.

Site-directed Mutagenesis

The wild-type bPRL phagemid was transformed into RZ1032 cells, and clones were selected by ampicillin resistance. A colony was grown in LB media, and a portion was transferred to 2 times TY media (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl in 1 liter of water) containing ampicillin and grown to a density of 0.3 at A. The culture was infected with R408 helper phage at a multiplicity of infection of 20. Cells containing both vectors were selected by resistance to kanamycin. After an overnight growth, the single-stranded uridine-substituted phagemid was purified from the media(26) .

Primers were designed to mutate serine 26, 34, or 90 to glutamic acid and to add a unique restriction site (BglII, EcoRI, and AvaI for S26E, S34E, and S90E bPRL, respectively). In vitro mutagenesis was performed as described by Kunkel et al.(26) using T7 DNA polymerase (catalog number 70017, U. S. Biochemical). The reaction products were transformed into DH5alpha cells and grown on LB agar plates containing ampicillin. Several colonies for each mutant were grown in LB media and subsequently analyzed by restriction digests. Colonies that were cut by the enzyme for the restriction site added during mutagenesis were selected, and the mutation was confirmed by sequence analysis.

Production and Purification of Recombinant Bovine Prolactins

Phagemids containing the wild-type or the S26E, S34E, and S90E mutants of DNA coding for methionyl-bPRL were transformed into BL21 cells, and positive clones were selected. One liter LB media cultures were grown to an optical density of A = 0.3. Protein expression was induced by the addition of isopropyl-beta-D-thiogalatopyranoside to 0.4 mM, and incubation of the culture was continued for 4 h. The cells were collected, resuspended in 100 mM Tris, pH 7.5, containing 1 mM phenylmethylsulfonyl fluoride and 25 mM dithiothreitol. Cells were broken by two passages through a French pressure cell. The inclusion bodies were collected by centrifugation, resuspended in 4.5 M deionized urea, 100 mM Tris, pH 11.3, and dissolved for 2 h at room temperature. The solubilized protein was incubated at 4 °C for 2 days and then dialyzed into 20 mM Tris, pH 7.4. The bPRLs were purified on a disposable DE52 anion exchange matrix (Whatman, Clifton, NJ) developed with a 0-750 mM NaCl gradient in the same buffer. The proteins were dialyzed into 5 mM ammonium bicarbonate and lyophilized. Each of the proteins was prepared under identical conditions.

Characterization of Recombinant DNA and Proteins

The pT7-7 phagemid DNAs containing the wild-type or the S26E, S34E or S90E mutations were each sequenced from primers annealing to phagemid sequences surrounding the polycloning region. The reactions used fluorochrome-labeled termination bases with T7 DNA polymerase (Sequinase, U. S. Biochemical), and the separation and data acquisition were performed by a fluorescence DNA sequencer (Applied Biosystems model 373A, Foster City, CA). Two reactions, reading opposite strands, sequence the 604-base pair insert.

The recombinant proteins were characterized for purity and size by SDS-containing 12% acrylamide gel electrophoresis under reducing conditions and by matrix-assisted laser desorption ionization-time of flight mass analysis using an external standard (model G2025A, Hewlett-Packard, Palo Alto, CA). An extinction coefficient was measured by absorption at 280 nm in 10 mM ammonium bicarbonate and related to the absolute amount of protein determined by quantitative amino acid analysis(27) . Absorption, fluorescence, and far UV circular dichroism spectra were measured at 22 °C for each recombinant bPRL.

Nb2 Rat Lymphoma Bioassay

The biological activity of each bPRL was measured in the Nb2 rat lymphoma biological assay(18) . Nb2 cells were grown in Fischer's medium containing penicillin (50 units/ml), streptomycin (50 µg/ml), 0.1 mM 2-mercaptoethanol and supplemented with 10% horse serum and 10% fetal bovine serum. The cells were grown in reduced serum (10% horse and 1% fetal bovine serum) for the 24 h immediately preceding the assay.

Stock solutions of recombinant bPRLs were prepared, their optical densities were measured at 280 nm, and their protein concentrations were determined by the bicinchoninic acid/copper sulfate assay(28) . The bPRL preparations were diluted in medium and added to 20,000 Nb2 cells in triplicate wells of 96-well culture plates at final concentrations of 0-10 ng/ml. The cells were incubated for 48 h. At the end of the growth period each well received 20 µl of Alamar Blue (Alamar, Sacramento, CA) and was incubated for an additional 4 h at 37 °C. The data provided by this vital dye method were highly correlated to cell counts and demonstrated a reduced variance within replicate determinations.

At the completion of the incubation the difference in absorbance of each well was measured at 570 and 600 nm. The difference was used to calculate an ED by a four-parameter fit method (ALLFIT program of Munson and Rodbard(29) ). The concentrations of the bPRLs were corrected by their extinction coefficients.


RESULTS

Characterization of the Recombinant DNA and Proteins

The wild-type bPRL phagemid DNA insert coded for a methyionyl residue followed by the complete DNA sequence of mature bPRL (30) . Each of the mutants was completely sequenced, and each contained the sequence appropriate for a silent restriction site and serine to glutamic acid mutation.

The proteins were expressed and purified with yields of final product varying between 3 and 12 mg/liter of fermentation. Correlation coefficients varied between 0.86 and 0.91 when the moles of the individual amino acids of recombinant or National Hormone and Pituitary Program bPRLs were correlated with the molar ratios calculated from sequence(30) . The recombinant proteins were greater than 95% homogeneous as observed on SDS-containing polyacrylamide gel electrophoresis under reducing conditions (Fig. 1). The molecular weights as determined by mass spectrometry were similar to the calculated values considering the precision (0.1%) and accuracy (0.1%) of the measurement. No contaminating proteins were observed in the mass spectra.


Figure 1: SDS-gel electrophoresis of recombinant bovine prolactins. 10 µg of each bPRL were run on a 12% polyacrylamide gel under reducing conditions. NIH, NIH bPRL; WT, wild-type bPRL; S26E, S26E bPRL; S34E, S34E bPRL; S90E, S90E bPRL.



Spectroscopy of the Recombinant Proteins

The concentrations of the wild-type, S26E, S34E, and S90E bPRLs used to measure circular dichroism spectra were determined by applying the molar extinction coefficients to the absorbance values. S26E, S34E, and S90E bPRLs had molar elipticities at 222 nm of 90, 94, and 86%, respectively, when compared with that of the wild-type bPRL (Fig. 2); these data suggest small reductions in helical contents resulting from the glutamic acid mutations. The 208 nm elipticity of the S90E bPRL is increased relative to the wild-type bPRL, suggesting a reduction in helical content.


Figure 2: Circular dichroism spectra for recombinant bovine prolactins. Proteins were in 20 mM NaPO(4) buffer, pH 7.5, with 150 mM NaCl. Concentrations determined by the extinction coefficients and the 280 nm absorbance were between 4.9 and 8.8 uM. Spectra were obtained at room temperature with a 0.0009684-cm pathlength after calibration with camphosulfonic acid in a Jasco Model J-500A spectropolarimeter.



The shape and maxima of the fluorescence spectra (excitation 280 nm) (Fig. 3) were similar for the wild-type, S26E, and S90E bPRLs, suggesting that water exposure of the tryptophane residues were either not changed by a mutation or were quenched by a mutation in the proximity of the tryptophane (Trp and S90E). In contrast, S34E bPRL displayed an 8.5 nm red shift (at half-peak height), indicative of a greater water exposure of the tryptophane residues.


Figure 3: Fluorescence spectra for recombinant bovine prolactins. Proteins were in 20 mM NaPO(4) buffer, pH 7.5, with 150 mM NaCl. The emission spectra were measured with a 280 nm excitation in a Perkin-Elmer model LS-50B fluorimeter at ambient temperature. The raw data (inset) were normalized as a fraction of the maximum intensity.



The UV absorbance spectra of wild-type and S90E bPRLs are nearly identical with maxima of 276 and 277 nm, respectively (Fig. 4). In contrast, the mutation of serines 26 or 34 produce significant blue shifts with small shifts of the maxima at 275 and 274 nm, respectively. The aromatic peak of the S34E bPRL is approximately 50% broader than the peaks of the other recombinant bPRLs.


Figure 4: UV absorbance spectra for recombinant bovine prolactins. Proteins were in 20 mM NaPO(4) buffer, pH 7.5, with 150 mM NaCl. The absorbance spectra were measured in a Uvicon Model 930 spectrophotometer at ambient temperature. The raw data (inset) were normalized to 280 nm.



Biological Activity of the Recombinant Proteins

The ED values of the bPRL preparations were calculated from the Nb2 biological assay data (Fig. 5) without fixing any of the four variables of the curve-fitting method (minimum, maximum, slope, and ED). EDs of wild-type, S26E, and S34E bPRLs were 18.6, 16.3, and 18.8 pM, respectively. The coefficient of variation of these measurements averaged 11.8%. The biological activities of the recombinant bPRLs were slightly less than the biological isolate of National Hormone and Pituitary Program bPRL (ED = 7.0 pM).


Figure 5: Biological activity of NIH and recombinant bovine prolactins. Prolactins were placed in 10 mM NH(4)HCO(3), and the concentration was calculated using the extinction coefficient. The Nb2 bioassay was performed as described under ``Materials and Methods'' using a vital dye to measure relative cell numbers.



The biological activity of S90E bPRL was dramatically reduced. The calculated ED of S90E bPRL was 1578 pM with none of the variables fixed, but the accuracy of this value is difficult to assess because the doses used in the assay failed to induce a full biological response. When the maximum and minimum variables were set as parameters, the ED for S90E bPRL was 672 pM with an 11% coefficient of variation. The activity of S90E bPRL is similar to the activity of our biological isolate of phosphorylated bPRL (727 pM) in the Nb2 bioassay(15) .


DISCUSSION

Replacement of serine 90 in bPRL with glutamic acid produced a hormone that behaved similarly in the Nb2 biological assay to the biological isolate of phosphorylated bPRL. These results confirm our interpretation of previous sequence and stoichiometry studies (20) that demonstrated serine 90 to be the most frequently phosphorylated and probably responsible for the reduced biological activity of the phosphorylated hormone. The similar reductions of ED values of the S90E and phosphorylated bPRLs suggested that glutamic acid was fully capable of functionally replacing phosphoserine. The presence of a negative charge is the most striking common structural feature of these two preparations. We suggest that this charge induces changes in the bPRL structure that disallows the protein to productively engage the receptor. The mechanism by which this occurs remains to be elucidated.

The sites by which lactogenic hormones interact and activate the lactogenic receptor are projected to be on sections of PRL that are distal to serine 90(8, 12, 13) . Therefore, phosphorylation or glutamic acid mimicry of bPRL at serine 90 must transduce structural changes to distant receptor-binding determinants and induce a change in their spatial relationships that preclude their productive interaction with the PRL receptor.

Several sets of structural elements may be required for this mechanism to function. First, a kinase recognition site must be present. Second, a set of local residues may be required that would interact with the phosphoserine or glutamic acid in position 90 to induce a local change in structure. Finally, other structural features must be present that respond to local alterations of structure by affecting distal elements.

The sequence surrounding serine 90 is RSWNDP. If one assumes, by projecting from structures of related proteins(8, 31) , that this sequence is a helix with a proline-induced break, then an N+4 salt bridge between Arg and Asp stabilizes the helical structure just N-terminal to Pro. The introduction of a negative charge at Ser between Arg and Asp might disrupt the secondary structure in this region. This may account for the 14% reduction in helical content observed between the wild-type and S90E bPRLs. Further studies will be required to conclude if the introduction of charge in an N+4 salt bridge is the local mechanism of transduction.

In contrast, the S26E and S34E bPRLs had biological activities that were indistinguishable from the wild-type methionyl-bPRL. Both S26E and S34E bPRLs show evidence of tertiary structural disruption by absorption and fluorescence spectroscopy. These spectroscopic changes suggest that aromatic residue hydration is increased, but the structural preturbations are neither sufficient nor specific to affect the biological activity.

In conclusion, glutamic acid mutations of each of the serines that are phosphorylated in vivo produce structural changes in bPRL that are observed by one or more spectroscopic methods. Relative to wild-type bPRL, S26E and S34E bPRLs appear to produce the blue shift in the absorption spectrum seen with phosphorylated bPRL but do not affect biological activity, whereas S90E bPRL produces a reduction in biological activity equivalent to that observed with phosphorylated bPRL.


FOOTNOTES

*
This work was supported by Grants DK42604 and DK01989 from the National Institutes of Health. 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: The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210. Tel.: 614-292-9641; cbrooks@magnus.acs.ohio-state.edu.

(^1)
The abbreviations used are: PRL, prolactin; bPRL, bovine prolactin; hGH, human growth hormone; hPRL, human prolactin.

(^2)
F. C. Peterson and C. L. Brooks, unpublished data.


ACKNOWLEDGEMENTS

Bovine prolactin was provided by the National Hormone and Pituitary Program. We acknowledge and appreciate the contributions of Michael Zehfus and Mark Maciejewski of the College of Pharmacy for aid in obtaining and interpreting the circular dichroism data.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.