0013-7~~;/92/1312-0973$03.00/0 Endocrinology Copyright01992byI'he Endocrine

EVIDENCE ,J;unes

Vol. 131, No. 2 in U.S.A.

Society

Printed

FOR A RAPID A.

Rillemal,

'Department ZDepartment

of of

STIMULATION George

Physiology, Physiology,

S.

OF TYROSINE

Campbell',

KINASE

David

Wayne State University

of

ACTIVITY

M. Lawsonl,

University Michigan

School Medical

BY PROLACTIN Christi"

IN Nb

RAT LYMPHOMA

CELLS

Carter-Sue

of Medicine, School, Ann

540 Arbor,

E.

Canfield, MI 48109-0622

Detroit,

MI

48201;

ABSTRACT. Studies were designed to determine if the activation of tyrosine kinases may be involved in the signal transduction pathway for PRL. Tyrosyl phosphorylation of cellular proteins was evaluated by western blot analysis of Nb2 cell proteins employing a" antibody to phosphotyrosine. Physiological concentrations of ovine PRL (oPRL) had a pronounced effect on the tyrosyl phosphorylation of a 121 kDa protein. Increased tyrosyl phosphorylation of the 121 kDa protein was detectable with concentrations of oPRL as low as 0.5 "q/ml. Consistent with oPRL acting through a PRL receptor, hGH also stimulated tyrosyl phosphorylation of the 121 kDa protein when tested at concentrations between 5 and 20 "q/ml. In time course experxnents, Increased tyrosyl phosphorylation of the 121 kDa protein was apparent after a 5 min incubation with 20 "g/ml At higher concentrations of hGH (200 "g/ml), increased phosphoryhGH, and maintained for at least one h. lation of the 121 kDa protein was clearly evident after only 1 min, indicating that tyrosyl phosphorylation of cellular proteins is a" early event following liqand binding to the PRL receptor. Increased tyrosyl phosphorylation of proteins of 40, 90 and 55-65 kDa was also evident after incubation with hGH for 10, 10, and 60 ml" respectively. These flndings are consistent with PRL-dependent tyrosine kinase activation being an early and perhaps initiating event in the signal transduction pathway for PRL in Nb2 cells. INTRODUCTION

cultures in "growth medium" as described previously (8). Cells used for experimentation were cultured for 24 h I" stationary medium (growth medium without fetal calf serum and with 3s0 rather than 10% horse serum) and placed in fresh stationary medium prior to use (8). The effects of lactogenic hormones on the phosphorylation of tyrosyl residues of Nb2 cell proteins were assayed by a modification of the method of Kamps and Sefto" (15,16). After incubating with human GH (hGH, pituitary-derived, gift of Dr. J.L. Kostyo, University of Michigan) or ovine PRL (oPRL, NIH-P-S-17, National Hormone and Pituitary Program, University of Maryland School of Medicine) for the specified times at 37 C, the cells were washed twice at O-4 C with 10 ml of phosphate buffered saline containing 1 mM orthovanadate, the" boiled in lysis buffer (16) for 5 min. Cellular proteins (100 vg) were resolved by SDS-PAGE and transferred to nitrocellulose filters by electroblotting. The filters were probed with anti-phosphotyrosine antibody (4GlO mouse monoclonal, upstate Biotechnology Inc., Lake Placid, NY) for 4 h. Bound antibody was detected with the ECL antibody detection system (Amersham Corp., Arlington Heights, IL) followed by autoradiography. To identify the molecular size of the PRL receptor, Nbg cells were cultured for 90 mi" with 10 pCi/ml [1251] hGH (192 pCi/pg, prepared by the lactoperoxidase method) in the presence or absence of 10 wg/ml unlabeled oPRL. Cells were incubated dt 23 C for 15 min in 3 ml of phosphate buffered saline containing the cross-linking reagent disucclr~imidyl suberate (0.45 mq). After two washes in phosphate buffered saline, the cells were solubilized in lysis buffer (16) and the 1ysates were subjected to SDS-PAGE and autoradiography.

anterior pituitary hormone PRL, in The addition to its well known function as a regulator is an important effector of a wide of lactation, range of physiological processes (1). While it is known that the various actions of PRL are mediated through its interaction with specific cell surface the molecular mechanisms by which the receptors, PRL-receptor interaction initiates these diverse responses remains largely unknown. Recently, receptors for PRL were cloned from several sources (reviewed in 2). These receptors differing primarily in the are highly homologous, length of their cytoplasmic domain. The predicted structure of the PRL receptors, while yielding few clues as to possible signal transduction pathways bhat might be used by these receptors, showed them to be members of the recently described cytokine/ hematopoietic growth factor receptor superfamily Although relatively little is known about (2). signaling by this superfamily of receptors, tyrosine kinase activity is ligand-stimulated associated with several members (3). including the Leceptor for GH (4,5), the member most closely related to the cloned PRL receptors. The present studies were designed to determine if the interaction of PRL with its receptors also involve a stimulation of may tyrosine kinase activity. As a model system, we have employed Nb 2 node lymphoma cells which exhibit a mitogenlc response to PRL (6-8) and express an intermediate length form of the PRL receptor (9). When these cells are synchronized by serum depletion, PRL induces a num&r of rapid responses including a" increased Ca uptake a" activation of Na+ /H+ exchange (ll), a" (101, rate of increased RNA synthesis (12) and a" increased accumulation of mRNAs for actin, c-myc, interferon requlatory factor I, and ornithine decarboxylase (13,14). How PRL initiates these responses has not been established. MATERIALS ~-

in

Iowa

City

Fig. 1 shows the dose response for the effects of the lactogenic hormones oPRL and hGH on the phosphorylation of tyrosyl residues in Nbp cell proteins. The most striking effect is on a protein (~~121) having a molecular weight of approximately 121,000. After a 1 h culture at 37 C oPRL at concentrations between 0.5 and 20 "g/ml

AND METHODS

node lymphoma cells (from Nbz Beer, Cancer Control Agency, Vancouver, Columbia, Canada) were maintained as

Received

RESULTS

on April

17,

Dr. C.T. British suspension

1992.

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121. stimulated tyrosyl phosphorylation of pp HGH, which is known to express its effects through PRL receptors in Nb2 cells (2,6,9,17-19), also 121. stimulated tyrosyl phosphorylation of pp Consistent with its biological potency (20), higher concentrations of hGH than of oPRL were required to stimulate tyrosyl phosphorylation.

Prl (rig/ml)

GH (rig/ml) c

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25

5

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20

05

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20

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FIG. 2. Time course of hGH promoted tyrosyl phosphorylation of Nb2 cellular proteins. Nbp cells were incubated with (+) or without (-) 20 or 200 rig/ml hGH for the indicated times and western blotting was performed. The anti-phosphotyrosine antibody and horseradish peroxidase conjugated second antibody were used at dilutions of 1:2000.

FIG. tyrosyl

1.

Potency phosphorylation

of

hGH and oPRL for promoting of Nb2 cellular proteins. cells were cultured with the indicated Nb 2 concentrations of hGH or oPRL for 1 h. Cell lysates were prepared and subjected to western blot analysis. The anti-phosphotyrosine antibody and horseradish peroxidase conjugated second antibody were used at dilutions of 1:SOOO. Fig. the time course for the 2 shows stimulation of phosphorylation of tyrosine kinase substrates in Nbg cells. At 20 rig/ml, hGH effects on the 121 kDa protein were clearly apparent by 5 min, and the response was maintained for at least 1 h. When hGH at a concentration of 200 rig/ml was employed, increased tyrosyl phosphorylation of pp121 was clearly visible at 1 min, indicating that increased tyrosyl phosphorylation of cellular proteins is an early event following ligand binding to the PRL receptor. Increased tyrosyl phosphorylation of 90, 40, and 55-65 kDa proteins was also evident (Fig. 2) after incubation with hGH for 10, 10 and 60 min respectively. Results of studies carried out to identify the molecular size of the PRL receptor in Nb2 shown in cells are Fig. 3. Cross-linked [12' I]hGH-receptor complexes migrated as proteins with a molecular weight of 92,000. When the molecular weight of the hormone (22,000) is subtracted from that of the hormone-receptor complex (92,000) the molecular weight of the receptor is calculated to be 70,000, consistent with previously reported values range which between 55,000 and 90,000 (9,17-19). Specificity of the binding of [ 125 I]hGH to its receptor is demonstrated in Fig. 3 where the [125 I]hGHreceptor does not complex appear on gels containing proteins from cells incubated with excess unlabeled oPRL. Similar results were obtained when using [ 125 I]oPRL in place of (data not shown). I '251]hGH DISCUSSION These

studies

provide

the

first

evidence

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FIG. 3. Cross-linking of [i25 I]hGH in Nb cells. Nb2 cells were incubated with 10 uCi/ml [ 155 I] hGH minus (Panels A,B:I) or plus (Panel B:II) 10 rig/ml oPRL for 90 min. After cross-linking, the cellular were subjected to SDS-PAGE, extracts stained with Coomassie blue (Panel A), and subjected to autoradiography (Panel B). Protein content of the cell extracts was 1.71 )lg protein/@. that lactogenic hormones acting through a PRL receptor rapidly stimulate the phosphorylation of tyrosyl residues of specific proteins. This finding suggests the fascinating possibility that activation of a cellular tyrosine kinase may play an initiating or propagating role in the signal transduction pathway for PRL, although we cannot rule out that PRL inhibits a tyrosine phosphatase or alters substrate accessibility. The possible involvement of a tyrosine kinase in the signaling pathway for PRL in Nbp cells is further suggested from experiments in which genistein (an inhibitor of tyrosine kinases) at concentrations above 5 ug/ml impairs the PRL stimulation of mitogenesis (Fan and Rillema, manuscript in preparation). The fact that the PRL receptor migrates as a 70 kDa protein indicates that the most prominent phosphorylated protein (~~121) is not the PRL receptor itself, in contrast to what has been observed for receptors with intrinsic tyrosine kinase activity (21). Also consistent with tyrosine kinase activity not residing within the PRL receptor is the finding that the cDNA for a PRL receptor cloned from Nb2 cells does not contain a consensus sequence for an ATP binding

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site (9). The inability to detect increased tyrosyl phosphorylation of a 70 kDa phosphoprotein in response to PRL or hGH raises the possibility that the PRL receptor may not be a substrate of a PRL-stimulated tyrosine kinase(s). However, our data cannot rule out the alternative possibility cross-linked migrates that the receptor anomalously or that phosphotyrosine residues in the receptor are recognized poorly by the antithis phosphotyrosine antibody used in study. if the 121 kDa or other Specifically how and phosphorylated proteins are involved in PRL stimulation of cell division or other responses to PRL in the Nbp cells remains to be established, intensity of although the early appearance and raises the interesting possibility that PPI21 pp121 itself may be a kinase. Identification of a PRL-stimulated tyrosine kinase(s) and determination of the means by which formation of the hormone-receptor complex causes increased phosphorylation should provide substantial insight into signal transduction pathways for PRL receptors. It is of interest to note that GH, acting through a somatogenic receptor, promotes tyrosyl phosphorylation of 121, 90 and two approximately 40 Kd proteins (thought to be ERKs 1 and 2) in 3T3-F442A fibroblasts (4,16, GS Campbell and C Carter-S", manuscript in preparation). Since PRL stimulates the tyrosyl phosphorylation of similar sized proteins in Nb2 cells, these findings suggest that lactogenic (PRL) and somatogenic (GH) receptors may, at least in part, share signal transduction pathways. Further, these results add the Nbg cell PRL receptor to the growing list of cytokine receptors that effect an increase in tyrosyl phosphorylation, and lend support to the notion that tyrosine kinase activation is a common, perhaps initiating, aspect of signaling by the members of this diverse family of receptors. REFERENCES 1. Nicoll CS 1974 Physiological actions of prolactin. In: Greep RO, Astwood EB (eds) Handbook of Physiology, Vol IV, Sect 7, Pt 2, Amer Physiol Sot, Washington DC, pp 253-292 2. Kelly PA, Djiane T, Pastel-Vinay M-C, Edery M 1991 The prolactin/growth hormone receptor family. Endocrine Rev 12: 235-251 3. Morla AO, Schreurs J, Miyajima A, Wang JY 1988 Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines. Mol Cell Biol 8: 2214-2218 4. Foster CM, Shafer JA, Rozsa FW, Wang X, Lewis SD, Renken DA, Natale JE, Schwartz J, Carter-S" C 1988 Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes. Biochemistry 27: 326-334 5. Stred SE, Stubbart JR, Argetsinger LS, Shafer JA, Carter-S" C 1990 Demonstration of growth hormone (GH) receptor-associated tyrosine kinase activity in multiple GH-responsive cell types. Endocrinology 127: 2506-2516 6. Gout PW, Beer CT, Noble RL 1980 Prolactinstimulated growth of cell cultures established from malignant Nb2 rat lymphomas. Cant Res 40: 2433-2436 7. Shiu RPC, Elsholtz HP, Tanaka T, Friesen HG,

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Gout PW, Beer CT, Noble RL 1983 Receptoraction of prolactin in a rat mediated mitogenic lymphoma cell line. Endocrinology 113: 159-165 8. Ofenstein JP, Rillema JA 1987 Possible involvement of the phospholipases in the mitogenic actions of prolactin on Nb2 node lymphoma cells. Proc Sot Exptl Biol Med 185: 147-152 9. Ali S, Pellegrini I, Kelly PA 1991 A prolactin-dependent immune cell line (Nbn ) expresses a mutant form of prolactin receptors. J Biol Chem 266: 20110-20117 10. Buckley AR, Montgomery DW, Kipler R, Putnam CW, Zukoski CF. Gout PW, Beer CT, Russell DM 1986 Prolactin stimulation of ornithine decarboxylase and mitogenesis in Nb2 node lymphoma cells: the role of protein kinase C and calcium mobilization. Immunopharmacology 12: 37-51 11. Too CKL, Cragoe+ I?$, Friesen HG 1987 Amiloride-sensitive Na /H exchange in rat Nbp node lymphoma cells. Stimulation by prolactin and other mitogens. Endocrinology 121: 1512-1520 12. Rillema JA, Tarrant TM, Linebaugh BE 1989 the mechanism by Studies on which prolactin RNA and DNA synthesis in Nbz regulates protein, node lymphoma cells. Biochim Biophys Acta 1014: 78-82 Fleming WH, Murphy PR, Murphy LT, Hatton TW, 13. Matusik RJ, Friesen HG 1985 Human growth hormone induces and maintains c-myc gene expression in lymphoma cells. Endocrinology 117: 2547-2553 Nb2 14. Yu-Li LY 1990 Prolactin stimulates transcription of growth-related genes in Nb2 T lymphoma cells. Molec Cell Endocrinol 68: 21-28 15. Kamps MK, Sefton BM 1988 Most of the substrates of oncogenic viral tyrosine kinases can be phosphorylated by cellular tyrosine protein kinases in normal cells. Oncogene Res 3: 105-115 16. Campbell GS, Pang L, Miyasaka T, Saltiel AR, Stimulation by growth hormone Carter-S" C 1992 of MAP kinase activity in 3T3-F442 A fibroblasts. J Biol Chem 267: 6074-6080 Webb CF, Wallis A comparison of 17. M 1988 lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques. Biochem J 250: 215-219 Elberg G, Kelly PA, Djiane J, Binder L, 18. 1990 Mitogenic and binding properties Gertler A of monoclonal antibodies to the prolactin receptor in Nb2 rat lymphoma cells. J Biol Chem 265: 14770-14776 19. Too CL, Shiu RPC, Friesen HG 1990 Crosslinking of G-proteins to the prolactin receptor in rat Nb2 lymphoma cells. Biochem Biophys Res Commun 173: 48-52 20. Cameron CM, Kostyo JL, Rillema JA, Gennick SE 1984 Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action. Am J Physiol 247: E639-E644 21. Geahlen RL, Harrison ML 1990 Proteintyrosine kinases. In: Kemp BE (ed) Peptides and protein phosphorylation, CRC Press, Boca Raton, pp 239-253 ACKNOWLEDGEMENTS This work was supported by NIH grants HD06571 (to JAR) and DK34171 (to CC-S); GSC was supported by NRSA post-doctoral fellowship GM14099. The authors wish to thank WJ Paradee, BE Linebaugh, LJ Christian, and LM Stevens for their technical contributions.

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