Interferon a Induces Rapid Tyrosine Phosphorylation of the uau Proto-oncogene Product in Hematopoietic Cells*

The uav proto-oncogene product (~95~"") is specifi- cally expressed in cells of the hematopoietic system, contains one Src homology 2 and two Src homology 3 domains, and is a substrate for receptor and non-recep-tor tyrosine kinases. Immunoblotting experiments using an anti-phosphotyrosine monoclonal antibody showed that interferon a (IFNa) induces rapid tyrosine phos- phorylation of ~95"" after binding to its cell surface receptor in the U-266 human myeloma cell line. The IFNa-induced tyrosine phosphorylation of ~95""" was time- and dose-dependent, confirming the specificity of the process. IFNa-dependent tyrosine phosphorylation of p95""" was also observed in other hematopoietic cell lines of B-cell origin (Daudi), T-cell origin (MOLT-8, and promyelocytic origin (HL-Bo). Immunoprecipitation ex- periments performed with SaP-labeled U-266 cells and phosphoaminoacid analysis of the bands corresponding to p9Sua" showed that ~95""" is phosphorylated on serine residues prior to IFNa stimulation of the cells. After IFNa stimulation significant amounts of phosphorylation of p95""" on tyrosine residues were detectable. "y- rosine phosphorylation of p9Sua" in U-266 and HL-Bo cells was also

p95""" has been shown to undergo transient tyrosine phosphorylation in T-cells during activation of the T-cell receptor-CD4 complex (5, 6), in activated mast cells (61, in cells transiently expressing epidermal or platelet-derived growth factor receptors in response to receptor activation (5, 6), in B-cells activated by engagement of their immunoglobulin M receptors (7), and in stem cell factor (c-kit ligand)-dependent human cells in response to stem cell factor activation (8).
IFNa' has been shown to transduce signals by inducing tyrosine phosphorylation of three components of the interferonstimulated gene factor 3 (ISGF3a), leading to the formation of the ISGF3a complex (9-11). ISGF3a translocates to the cell nucleus and in association with ISGF3r binds to a DNA sequence, the interferon-stimulated response element, to initiate IFNa-dependent gene transcription (Sll). It has also been shown that an early event in the IFNa signal transduction pathway is tyrosine phosphorylation of the a-subunit of its receptor (12). However, in some cells expressing the variant form of the IFNa receptor, IFNa response can be elicited in the absence of phosphorylation of the a-subunit (13). DNAcomplementation studies have shown that a tyrosine kinase involved in the IFNa signal transduction pathway is the tyrosine kinase p135LYk2 (14). We have recently observed that IFNa induces tyrosine phosphorylation of multiple proteins in addition to the p135'yk2 kinase and the ISGF3 components in hematopoietic cells.2 As the proto-oncogene product ~95""" has been shown to be a substrate for receptor and non-receptor tyrosine kinases, and possibly link surface signals with downstream cellular events, we sought to determine whether ~95""" is among the substrates phosphorylated in response to IFNa. Our findings suggest that p95""" may be part of a n IFNa-signaling pathway in cells of hematopoietic origin.

EXPERIMENTAL PROCEDURES
Cell CuZturesThe human myeloma U-266 cell line and the HL-60 acute promyelocytic leukemia cell line were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.) and antibiotics. The human lymphocytic cell lines Daudi (Burkitt's lymphoma) and MOLT-4 (acute T-cell lymphocytic leukemia) were grown in RPMI 1640 supplemented with 10% (v/v) defined calf serum (HyClone Laboratories, Logan, U T ) and antibiotics.
Labeling of Cells with P2P10rthophosphoricAcid-Cells (0.5-1 x 109 were washed three times with phosphate-free RPMI 1640 and cultured for 30 min a t 37 "C in phosphate-free medium. The cells were subsequently incubated for 3 h in phosphate-free medium with carrier-!?eeP*P]orthophosphoric acid (Du Pont NEN) a t a concentration of 0.1 mCi/ml. The labeled cells were subsequently stimulated with lo4 units/ml IFNa for the indicated times and lysed in phosphorylation lysis buffer. The lysates were immunoprecipitated with the indicated antibodies, washed five times in phosphorylation lysis buffer, and analyzed Phosphwmino Acid Analysis-Phosphoamino acid analysis was performed as previously described (12, 15). Briefly, "P-labeled proteins were transferred to polyvinylidene fluoride membranes (Immobilon, Millipore). The membranes were rinsed three times with deionized water, dried, and subjected to autoradiography. After identification of the phosphorylated proteins, the pieces of the membrane containing the individual phosphoproteins were cut out and rewetted sequentially with methanol and deionized water. The peptides were subsequently hydrolyzed in 200 pl of 6 N HCI a t 110 "C for 1 h. The acid was separated from the Immobilon by centrifugation, and samples were lyophilized twice. Samples were subsequently resuspended in pH 3.5 buffer (glacial acetic acid:pyridine:water, 5:0.5:94.5, v/v/v), and the individual phosphoamino acids were separated by electrophoresis in pH 3.5 buffer at 1 kV. Control phosphoseriqe, phosphothreonine, and phosphotyrosine were detected by reaction with ninhydrin, and radioactive phosphoamino acids were detected by autoradiography.
by SDS-PAGE.

RESULTS
Tyrosine Phosphorylation of ~95""" by Type Z ZFNs-'Ib determine whether p95""" is a substrate for IFNa-dependent tyrosine phosphorylation, U-266 cells were treated with IFNa2 for different periods of time, the cells were lysed, and the lysates were immunoprecipitated with either control normal rabbit serum or a polyclonal antibody against p95""", prior to SDS-PAGE analysis and anti-phosphotyrosine immunoblotting. Fig.   LA shows that IFNa induced tyrosine phosphorylation of ~9 5 "~" within 1 min after binding to its receptor, and the signal gradually declined after 60 min of IFNa treatment. Weak base-line tyrosine phosphorylation of p95"" (prior to IFNa stimulation) was detectable after longer exposure of this and other blots. Reprobing of the same blot with the anti-p95""" polyclonal antibody confirmed that equal amounts of p95""" were present in the immunoprecipitates of IFNa-untreated and treated cells (Fig. 1 B ) . When cell lysates of IFNa-treated cells were immunoprecipitated with the anti-p95""" antibody in the presence of the immunizing peptide, the appearance of the 95-kDa band detected in anti-phosphotyrosine immunoblots was blocked, confirming that it corresponds to the proto-oncogene product ~95""" (data not shown). A dose-response experiment of the IFNa-induced tyrosine phosphorylation of ~95""" in U-266 cells is shown in Fig. 2. IFNa induced tyrosine phosphorylation of p95""" in a dose-dependent manner, further indicating the specificity of the process.
We subsequently studied whether IFNa induces tyrosine phosphorylation of ~95""" in other cell lines of hematopoietic origin. IFNa-dependent tyrosine phosphorylation of ~95""" was also detected in cells of promyelocytic origin (HL-601, T-cell origin , and B-cell origin (Daudi) (Fig. 3). Significant amounts of base-line tyrosine phosphorylation of p95""" were detected in Daudi and MOLT-4 cells, but IFNa significantly increased the tyrosine phosphorylation of ~95""" (Fig. 3). Tyrosine phosphorylation of ~95""" in U-266 and HL-60 cells was also observed when cells were treated with IFNP or IFNo, suggesting that ~95""" may be involved in the signaling pathway of all Type I IFNs (Fig. 4).
32P Labeling and Phosphoamino acid Analysis-We next sought to determine whether p95""" is also phosphorylated on serine andlor threonine residues in the human U-266 myeloma cell line. Cells were labeled with [32P]orthophosphate, and the phosphoaminoacid content of ~95""" was examined before and IFNa-dependent tyrosine phosphorylation of p 9 5 " . U-266 cells were stimulated with lo4 unitdm1 IFNa2 for the indicated times a t 37 "C. Cell lysates were immunoprecipitated with either control rabbit serum (lanes 1 3 ) or an antibody against ~95""" (lanes 4
after IFNa stimulation of the cells. Fig. 5A shows that ~95""" is phosphorylated prior to IFNa treatment, and that its phosphorylation increases significantly upon IFNa stimulation of the cells. Phosphoamino acid analysis of the bands corresponding to ~95""" showed a significant amount of phosphorylation on serine residues prior to IFNa treatment, with only trace amounts of phosphorylation on tyrosine residues. After IFNa treatment of the cells significant amounts of phosphorylation of ~95""" on tyrosine residues were detectable (Fig. 5B), confirming the immunobloting findings.

DISCUSSION
The 'Type I IFNs have pleiotropic biological effects that include immunoregulation, antiviral activity, and anti-proliferative effects on normal and neoplastic cells (16). The mechanisms, however, by which Type I IFNs induce their effects are not completely understood. Recently, it has been shown that tyrosine phosphorylation can directly activate an IFNa-dependent latent transcription factor (9-11), suggesting that second messengers may not be necessary for the initiation of transcription of known interferon-stimulated genes (9-11). The tyrosine kinase has been shown to associate with the a-subunit of the IFNa receptor and to be activated by IFNa (17), suggesting that it may be the kinase regulating the phosphorylation of the ISGF3 components. In the current study we sought to determine whether ~95""" is a substrate for IFNadependent tyrosine kinase activity in the U-266 human myeloma cell line. Immunoblotting experiments disclosed that p95""" is phosphorylated on tyrosine in a time-and dose-dependent manner in response to IFNa. The immunoblotting experiments were subsequently confirmed by 32P labeling experiments and phosphoaminoacid analysis of the immunoprecipitated p95"" protein. Tyrosine phosphorylation of p95"" was also inducible by two other Type I IFNs, IFNP and IFNo. Taken together these data suggest that p95""" may be involved in a Type I IFN-dependent tyrosine kinase signaling pathway. The biological significance of the IFNa-dependent tyrosine phosphorylation of ~9 5 "~" , however, remains to be determined. Three of the cell lines studied here Daudi, have been shown previously to be sensitive to the anti-proliferative effects of IFNa (13,18,19). HG60 cells, in which we also demonstrated phosphorylation of ~95""" by IFNa and IFNP, do not respond directly to the anti-proliferative effect of IFNa (19,20). However, it has been shown that in these cells IFNa and IFNP act synergistically with phorbol esters and retinoic acid to induce differentiation (20, 21). Interestingly, in these cells tyrosine phosphorylation of ~95""" increases dramatically when cells differentiate to neutrophils in response to retinoic acid (22). Based on these observations and our findings, it is tempting to hypothesize that ~95""" may be involved in a 'Type I IFN-signaling pathway regulating hematopoietic cell differentiation. Such a hypothesis is further supported by the fact that ~95""" is specifically expressed in cells of hematopoietic origin; therefore, the pathway in which it may be involved should mediate signals specific for the effects of IFNa in these cells.
~95""" contains regions of homology with the bcr and dbl oncogenes and the yeast CDC24 proteins (4,26). It also contains zinc finger-like, helix-loop-helix-like, and leucine zipperlike domains (1)(2)(3). Because of these later motifs, it has been suggested that ~95""" may either act as a transcriptional activator or regulate the activity of transcription factors in a similar manner with other proteins containing these motifs (5, 6). Although there is not evidence at this time that p95""" binds DNA, it is conceivable that ~95""" may function as a transcrip-IFNa Induces Qrosine Phosphorylation of ~95""" tional activator of some interferon-stimulated genes. Recently, it has been also shown that p95""" has guanine nucleotide exchange activity in T-cells, that increases after T cell antigen receptor triggering, parallels its tyrosine phosphorylation, and may be regulating the activation of the ras oncogene (24). This raises the question of whether in response to IFNa, p95""" functions also as a regulator of guanine nucleotide exchange of ras-encoded proteins. Interestingly, it has been shown that double-stranded RNA induces expression of the c-Ha-ras gene in NIH 3T3 cells that is inhibited by anti-beta interferon antibodies, suggesting that IFNP may mediate the induction (25). Studies to determine the effect of IFNa and IFNp on the activation of ras-encoded proteins or other members of the ras superfamily on which p95"" may act as guanine nucleotide releasing factor (e.g. the rho subfamily) may be of interest and provide important information on the role of p95""" in IFNa signaling.
providing the IFNa2, Dr. Gary Williams (Berlex) for providing the