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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. RAPID COMMUNICATION Tee Protein-Tyrosine Kinase Is Involved in Interleukin-3 Signaling Pathway By Hiroyuki Mano, Yoshihiro Yamashita, Ken Sato, Yoshio Yazaki, and Hisamaru Hirai Among cytoplasmic protein-tyrosine kinases (PTKs) Tec now forms a novel subfamilywith recently identified Tec-related PTKs (Btk and ltk/Tsk). Tec is known to be abundantly expressed in myeloid cells, and multiple forms of Tec protein can be generated viathe mechanism of alternative splicing. In this report, we have inveatigated 5“terminal diversityof the tec messagesto demonstrate a predominant form of the Tec protein in mouse hematopoietic cell lines. Using anti- N ONRECEPTOR TYPE protein-tyrosine kinases (PTKs) can be divided into two groups, namely, the Src-family (Src, Yes, Fyn, Lyn, Blk, Lck, Hck, Fgr, and Yrk) and the non-Src family (Tec, Btk, Itkfhk, Fps/Fes, Fer, Abl, k g , Zap-70, Syk, Tyk-2, Jak-l, and Jak-2).’ Well-shared characters among the members of the Src-family are (1) an Nterminal myristylation site: (2) a C-terminal tyrosine residue as the negative regulatory site (corresponding to Tyr-527 in c - S r ~ ) and , ~ (3) Src homology (SH) 2 and SH3 domains in the noncatalytic region.“ In contrast to the conserved protein structure among the members of the Src family, the non-Src family members have rather divergent structure^.^.^ None of them has C-terminal phosphotyrosine-acceptor sites. Only type IV protein of murine c-Ab1 among them is presumed to be myristylated.’ Furthermore, the Jak kinases do not even have SH2 or SH3 domains: The Tec kinasewas introduced asa novel member of the non-Src family.’ Tec has SH2,SH3, and kinase domains as the members of the Src family.However,thereare no myristylation sites or C-terminalphosphotyrosine-acceptor sites in the predicted Tec protein. Recently, two Tec-related kinases have been reported by other groups. Siliciano et a19 have identified the Itkkinase that is inducibly expressed by interleukin-2 (IL-2) in T cells, and the same kinase was also reported by Heyeck and Berg” as Tsk. Tsukada et all’ and Vetrie et all2 have independently published another Tec-related kinase (Btk) responsible for X-chromosome-linked agammaglobulinemia (XLA). All three kinases(Tec, Itk/Tsk, and Btk) have thesame protein structure, and their amino acid sequences are exceptionallyhomologouscompared with the othermembers of nonreceptor PTKs. Therefore, these three kinases should constitute a novel subfamily among the nonreceptor type PTKs. All members of the Tec family are abundantly expressed in hematopoietic tissues. Because the Btk kinase plays a pivotal role in Ig production in B cells, the other members of the Tec family are expected to be directly involved in the signaling systems of either mitogenic pathways or cellspecific functions in the hematopoietic system. In contrast to the high expression of ItkrTsk in T cells and Btk in B cells, Tec is abundantly expressed in myeloid ~el1s.l~ Therefore, it has been an intriguing issue to investigate the in vivo role of the Tec kinase in myeloid cells. We describe here the detailed analysis of alternative splicing of the 5”region of the tec messages. Furthermore, by using anti-Tec serum, we could show that Tec is involved in the signaling pathway of IL-3. Blood, Vol 85, No 2 (January 15). 1995 pp 343-350 Tec serum,we could showthat stimulationwith interleukin3 (IL-3) can induce tyrosine phosphorylation of Tec both in IL-3 stimulation was also myeloid andpro-B-celllines. shown to inducekinase activity of Tec. Furthermore, we could demonstratethat Tec is constitutively associated with the Shc protein in vivo. Thus, we conclude that Tec is involved in the signaling pathway of IL-3. 0 l995 by The AmericanSociety of Hematology. MATERIALS AND METHODS Cells and culture. A myeloid cell line, 32D,I4 and a pro-B-cell line, BaF3,” were maintained in RPM1 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) and 25 U/mL of murine IL-3. For stimulation experiments, cells were depleted of L 3 overnight and then stimulated with L - 3 for the period of 5 minutes unless otherwise indicated. COS-l cellsi6 were maintained in Dulbecco’s modified Eagle’s medium (GIBCO) with 10% FCS. RNA-dependent polymerase chain reaction (PCR) amplifcation of the 5’-region of the tec messages. Primers of the inner set (sense,GCTCTAGATTGGCTTGTCTC;antisense,TCGGTACCTGCTTTGTGGA) and the outer set (sense, GCAGTTTGGACGTCGCTC; antisense,AACCTTCTTCACCCATCGG) were synthesized by the model 381A DNA synthesizer (Applied Biosystems, Foster City, CA). From 1 pg of total RNA of 32D cells, the first strand of the tee cDNA was synthesized with the antisense primer of the outer set using murine reverse transcriptase (SuperScript; BRL, Gaithersburg, MD) according to the manufacturer’s instructions. The same reaction without reverse transcriptase was used as the negative control for further experiments. One twentieth of the cDNA products was PCR-amplified by the primers of the outer set at the cycle of 92°C for 1 minute, 37°C for 1 minute, and 72°C for 2 minutes for 30 courses using AmpliTaq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT) according to the manufacturer’s protocol. Onefiftieth of the first PCR-product was further PCR-amplified by the primers of the inner set at the same condition for 30 cycles. Recognition sequences of the restriction enzymes, Xba I and Kpn I, were incorporated at the ends of the sense primer and the antisense primer of the inner set, respectively, to facilitate subcloning. From the Department of Molecular Biology, Jichi Medical School, Yakushiji, Minami-Kawachi-machi, Kawachi-gun,Tochigiken, Japan; and The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. Submitted July 21, 1994; accepted October 24, 1994. Supported in part byGrants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture and from the Ministry of Health and Welfare, Japan. Address reprint requests to Hiroyuki Mano, MD, PhD, Department of Molecular Biology, Jichi Medical School, 331 1-1 Yakushiji, Minami-Kawachi-machi, Kawachi-gun, Tochigi-ken 329-04, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 I995 by The American Society of Hematology. 0006-4971/95/8502-0036$3,00/0 343 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. MAN0 ET AL 344 SH-3 SH-2 Kinase domain Type 1 Type 1 l l I l \ I , \ l ! I Fig 1. Comparison the of predicted structure among Tec types 1, II, 111, and IV. Structures of the Tec kinase are aligned. Divergent regions amongTec (l, subtypes generated alternative splicing by of Tec messages are differently shaded. SH2, SH3, and kinase domains are also indicated. l l Type DNA sequencing of rhe PCRproducrs. The PCR products of 475-bp and 388-bp length were separated through 2 8 agarose gel (NuSieve 3:1 Agarose: FMC BioProducts, Frederick, MD) and purified by the Geneclean kit (Bio 101, Inc. La Jolla, CA). Each fragment was then subcloned into Bluescript phagemid vector (Stratagene, La Jolla, CA). Double-stranded DNAs of the recombinant plasmids were denatured and sequenced by the chain-termination method" using the T7 or the T3 primers (Stratagene). Prepararion of anti-Tec serum. The cDNA fragment encoding the SH3 domain of mouse Tec type IV* (see Results) was PCRamplified and subcloned into the pGEX-3X vector (Pharmacia LKB Biotechnology, Uppsala. Sweden). NMS22 cells (Stratagene) expressing this plasmid were grown i n LB medium containing S0 p'plmL ampicillin and stimulated with 0. I mmolL isopropyl-0-D*The amino acid sequences of Tec types 11, 111, and IV have been deposited in the NRRF-PIR data base under accession numbers JU021S. 3110227. and 3110228, respectively. A Protein IA (87 bp) - 5' \ +G\ /J /I /' /' 5' +i+ U IIA (41 bp) cDNA cDNA I Protein " B 2036 + 1636 + 1018 + Fig 2. (A) Alternative splicing of 5"region of the tec messages. Thin linesrepresent cDNAs of rectype Isand type11.'' Nucleotide sequences specific t o each type (IA or IIA sequences) are emphasized as thick lines. Predicted N-termini of the Tee proteins are illustrated as boxes, aligned t o their corresponding cDNAs. Divergent amino acid sequences between type I and type II are differently shaded. Nucleotide sequences used for primers in RNA-dependent PCR amplification (see Materials and Methods) are delineated as arrows. (B) RNA-dependent PCR products of the rec5"region. Sizes of molecular weight markers (l-kb ladder; BRL) are shown as basepairs at the left side of the panel. The part of tec cDNA surrounding the IA and IIA sequences was PCR-amplified from thereaction of cDNA synthesis with I + ) or without (-1 reverse transcriptase (see Materials and Methods). The predominant 475-bp and 388-bp PCR products are indicated by arrows. 517/506+ 396 + 344 -g 298 220 -b +475 +388 bp bp From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 345 TECIS INVOLVED IN IL-3 SIGNALINGPATHWAY thiogalactopyranoside (IPTG) for 3 hours. The fusion proteinof Tec SH3 and glutathione S-transferase (GST) was purified through glutathione-Sepharose 4B column (Pharmacia LKB Biotechnology) according to the method of Smith and Johnson.’*The purified fusion protein wasmixedwith complete Freund’s adjuvant (Sigma, St Louis, MO) and injected into rabbits to raise anti-Tec serum against its SH3 domain. For boosting, the protein was mixed withincomplete Freund’s adjuvant (Sigma). Transient expression in COS-I cells. The tec type IV cDNA was subcloned into an expression vector, pSSRal’ to generate the pSSRa/tec plasmids. COS-l cells ( 5 X IO’ cells/experiment) were transfected with 1 pg of pSSRa or pSSRaltec by the diethyl aminoethyl (DEAE)-dextran method?” After 72 hours of culture, cells were lysed in the NP-Lysis buffer (50 mmol/L HEPES, pH 7.4, 0.5% Nonidet P-40, 150 mmol/L sodium chloride, 1 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate. 200 U/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride). After incubation for 30 minutes on ice, cell extracts were clarified by the centrifugation of 10,OOOg for 10 minutes. Immunoprecipitation and in vitro kinase assay. Cell lysates of 32D or BaF3 cells (1 X IO’ cells/experiment) were prepared as described above. For immunoprecipitation, equal amounts of cellular proteins were mixed with either normal rabbit serum, anti-Tec serum (1:400 dilution for each), or anti-Shc antibody (Upstate Biotechnology Inc. Lake Placid, NY) for 2hours at 4°C. Lysates were incubated with protein A-Sepharose 4B beads (Sigma) on a rotating platform for another 2 hours. The immunecomplexes were extensively washed with the lysis buffer and boiled in Laemmeli’s sample buffer” for 3 minutes. For in vitro kinase assay, the immunecomplexes were washed three times with the NP-lysis buffer, washed three times with the kinase buffer (20 mmol/L HEPES, pH 7.4, 150 mmol/L NaCI, IO mmol/L MgClz, IO mmol/L MnCI,), and finally incubated with 0.37 MBq of [y-”P]ATP (Amersham, Arlington Heights, IL) for 15 minutes at room temperature. Imrnunohlotring. Twenty micrograms of cellular proteins or one third of the immunecomplexes were separated through 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)” and electroblotted onto polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA) by the standard procedures.” The membranes were incubated at 4°Covernight in TBST (20 mmoll L Tris-HCI, pH 7.4, 150 mmollL NaCI, 0.05% Tween 20) with 2% bovine serum albumin (fraction V; Sigma). The membranes were incubated with anti-Tec serum (1:10,000) or antiphosphotyrosine antibody (PY 20; ICN Biomedicals, Irvine, CA) at room temperature for 1 hour in TBST. After extensively washed with TBST, the membranes were subsequently incubated with protein A/G conjugated with horseradish peroxidase (Pierce, Rockford, IL) at room temperature for 1 hour in TBST and specific bindings were visualized by the ECL detection system (Amersham) according to the manufacturer’s instructions. Metabolic laheling and phosphoamino acids analysis. For [‘*P]orthophosphate labeling, 32D cells (1 X IO’lexperiment) were cultured in phosphate-free RPM1 1640 medium (GIBCO) supplemented with 37 MBqlmL of [”Plorthophosphate (Amersham) without IL-3 or FCS for 2 hours and then stimulated with IL-3 for 5 minutes. Tec was immunoprecipitated from the lysates of these cells, separated through 7.5% SDS-PAGE, and electroblotted onto PVDF membranes as described above. After taking autoradiography, parts of the membrane containing the Tec proteins were excised and boiled in 5.7 N HCI according tothemethodofKampsand Sefton.” Phosphoamino acids were separated by one-dimensional electrophoresis at pH 3.5 on a cellulose thin-layer plate.” RESULTS Alternativesplicing of the tec messages. After first reporting the rec cDNA,” we could obtain other cDNAs that 205 117 -b 80 -W +Tec Fig 3. Tec type IV is a peptide of 70 kD in vivo. Total cell lysates of COS-l cells (2 pgllane) transfectedwith either pSSRa vector alone (Vector) or the tec-expressionconstruct (tecl or total celllysates of 32D cells 120 p g ) (32D) were subjected to 7.59/0 SDS-PAGE and immunoblotted with anti-Tec serum. Theposition of Tec is indicated by an arrow. The positionsof molecularweight standards are shown at the left (xlo-’)). can encode different forms of Tec proteins.” The original cDNA (type l)* and the recently identified one (type were generated from alternatively spliced tec messages and can encode different sets of N- and C-termini of the Tec proteins (Fig 1). Each cDNA of type I and I1 has a specific insertion of nucleotides at its 5”region (shown as l-A or IIA sequences in Fig 2A), resulting in alteration of the Ntermini of the predicted proteins. However, detailed analyses including the molar ratio of the two insertions have not been performed. To show the predominant N-termini of Tec in hematopoietic cells, the tec cDNA fragments encompassing the two insertion points were PCR-amplified by the nested primer sets (denoted by arrows in Fig 2A). As shown in Fig 2B, two major PCR products of 475-bp and 388-bp length From www.bloodjournal.org by guest on June 18, 2017. For personal use only. MAN0 ET AL 346 A B 205 aP-Tyr +Tec - 80 - 116 aP-Tyr +Tee 50 0 IC aTec 1 5 aTec 10 20 C aP-Tyr aTec Fig 4. Induction of tyrosine phosphorylation of Tec. (A) Total cell lysates (20 pg/lane) and immunoprecipitates of normal rabbit serum (NRS)or anti-Tec serum (aTec) from 32D cells (1 x 10’ cellslexperiment) with (+) or without (-1 11-3 stimulation (100 UlmL for 5 minutes) were separated through 7.5% SDS-PAGE and subjected t o immunoblotting with antiphosphotyrosine antibodylaP-Tyr). The same filter was reprobed with anti-Tec serum (crTec) t o compare the amounts ofTec loaded. The position of Tee is indicated by anarrow. (B) The same set of experiment as in (AI were performedin BaF3 cells. The positions of molecular weight standards are shown at theleft (xlo-’)). (C) 32D cells (1 x lo7 cellslexperiment) were starved of IL-3 overnight and restimulated with IL-3 (100 UlmL) for 0, 1, 5, 10, or 20 minutes as indicated at the top. Tec was immunoprecipitated from each cell lysate and was subjected t o immunoblotting with antiphosphotyrosine antibody (aPTyr). The same filter was reprobed with anti-Tec serum (crTec). were amplified in equal amounts. Because the two products could notbe generated from the reaction without reverse transcriptase, they were not synthesized from contaminated genomic DNAbut rather from RNA. DNA sequencing of the two fragments has shown that the 475-bp product contains both l-A and 11-A insertions (designated as the IA+/ IIA+ fragment), whereas the 388-bp product contains only 11-A (designated as the IA-/HA+ fragment). Based on nucleotide sequences, tec cDNAs containing either the IA+/ IIA+ fragment or the IA-/HA+ fragment should use the same translation start site used for Tec type Therefore, the predominant N-terminus of the Tec kinase should be the same as that of Tec type 11. As previously reported, the predominant C-terminus of the Tec kinase is the same as that of Tec type 1.l3 Thus, Tec proteins with the type II-N- terminus and the type l-C-terminus should be predominant. We could observe that Tec proteins of this form are predominant not only in hematopoietic cells but also in liver (data not shown). In addition, two forms of the Tec SH3 domain have been reported to be generated by alternative splicing of the tec messages.” In conclusion, there should betwo major forms of the Tec kinase in vivo; both have the same type 11-N-termini and the type I-C-termini, but differ in the SH3 domain. We designate them, respectively, as Tec type I11 (with the truncated SH3 domain) and type IV (with the full SH3 domain), whose predicted molecular weights are 71 116 and 73604, respectively (Fig 1). In the case of 32D and BaF3 cells, PCR analysis of the SH3 domain of the tec messages could identify only the long form of the SH3 domain (type IV) but not the short one (type 111) (data not From www.bloodjournal.org by guest on June 18, 2017. For personal use only. TEC IS INVOLVED IN IL-3 SIGNALING PATHWAY shown). Thus, type IV should be the predominant form of Tec in these cell lines. IL-3 caninducetyrosine phosphorylation of Tec. First, anti-Tec serum was verified using COS-l cells expressing the Tec protein. Total cell lysates of 32D cells and COS-l cells transfected with either pSSRa alone or pSSRa/tec (type IV cDNA) were immunoblotted with anti-Tec serum. As shown in Fig 3, anti-Tec serum could recognize a peptide of approximately 70 kD specifically in 32D cells and the tec-transfected COS-l cells, but not in the vector-transfected A 200 + 117+ 80 + 50 + B ( 4 . ' 2 . cells. This peptide could not be detected in the lysate of 3T3 cells that do not express the tec message endogenously (data not shown). Therefore, we conclude that Tec type IV is a peptide of 70 kD in vivo. A peptide of approximately 80 kD observed in Fig 3, lane 32D, is of the nonspecific binding of protein NG-horseradish peroxidase, because this band could not be observed when a different detection system with alkaline phosphatase was used (data not shown). To analyze the role of Tec in the IL-3 signaling pathway, tyrosine-phosphorylation of Tec was investigated in an IL-3dependent myeloid cell line, 32D (Fig 4A). Total cell lysates, normal rabbit serum immunoprecipitates, and anti-Tec serum immunoprecipitates from 32D cells were immunoblotted with antiphosphotyrosine antibody (aP-Tyr part). IL-3 stimulation results in the induction of tyrosine phosphorylation of pp7OTCc. The same filter was reprobed with anti-Tec serum to compare the amount of the Tec proteins immunoprecipitated (aTec part). To show that Tec is involved in the IL-3 signaling system in lymphoid cells as well as in myeloid cells, the same set of experiments was performed in an IL3-dependent pro-B-cell line, BaF3. As shown in Fig 4B, Tec is also inducibly tyrosine-phosphorylated in BaF3 cells with the stimulation of IL-3. Next, the time-course of Tec phosphorylation by the IL-3 stimulation was investigated in 32D cells. As shown in Fig 4C, tyrosine phosphorylation of pp70T" reaches to the maximum at 1 to 10 minutes after the IL-3 stimulation and decreases thereafter. Phosphorylation level of Tec. To confirm the effect of L - 3 on Tec phosphorylation, Tec was immunoprecipitated from the lysates of 32D cells metabolically labeled with [32P]orthophosphateand was blotted onto PVDF membrane. An autoradiogram of the membrane shows that 5 minutes of IL-3 stimulation could induce phosphorylation of pp7OTec (Fig 5A). The parts of the membrane containing the Tec proteins were subsequently excised and boiled in 5.7 N HCI. One-dimensional electrophoresis of the acid-hydrolyzed peptides shows that Tec is faintly phosphorylated on serine and threonine residues in the absence of IL-3 (Fig 5B, -). In contrast, IL-3 stimulation could induce phosphorylation of all serine, threonine and tyrosine residues (Fig 5B, IL-3). IL3 can induce kinase activity of Tec. Tec was immunoprecipitated from 32D cells at various periods after L 3 stimulation and was subjected toin vitro kinase assay without exoge- 4 IL-3 8 347 p-Ser p-Thr Fig 5. Phosphoamino acids contents of Tec. (A) Tec lafec) was immunoprecipitatedfrom cell lysatesof 32D cells l1 x lo7cellslexperimentl metabolically labeled with 13'Plorthophosphatewith (IL-3) or without (-1 IL-3 stimulation (5 minutes) and was subjected to 7.5% SDS-PAGE. Normal rabbit serum immunoprecipitatesINRS) from the corresponding cells are also subjected to 7.5% SDS-PAGE. The proteins were electrotransferredonto PVDF membrane and autoradiographed. The positions of molecular weight standards are shown at the left IxlO-'). (B)The parts of the membrane containing the Tec protein immunoprecipitatedfrom 320 cells with IIL-31 orwithout (-1 the IL-3 stimulation were excised and subjected to thephosphoamino acidanalysis.Acid-hydrolyzedTee was separatedbyone-dimensional electrophoresis at pH 3.5 on a cellulose thin-layer plate. The positions of phosphoserine (p-Ser), phosphothreonine (p-Thr), and phosphotyrosine (p-Tyr) are indicated at the right. The regions of migration of the unlabeled phosphoamino acids aremarked by dotted lines. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. MAN0 ET AL 348 0 5 io 20 30 40 Fig 6. In vitro kinase assay of Tec. After starvation of IL-3.32D cells were stimulated with IL-3 for 0,5,10, 20,30, or 40 minutes as indicated at thetop. Tec was immunoprecipitatedfrom each fraction and subjected to in vitro kinase assaywithout exogenous substrates. The positions of Tec, p68, p63, and p58 are shown. noussubstrates.Asshown in Fig 6, stimulationwith L 3 induced a phosphorylation level of Tec (indicated by an arrow), p68, and p63. The kinase activity reached its maximum 5 to 10 minutes after the IL-3 stimulation. Faint phosphorylationof another cellular protein (p58) can be recognized when the Tec kinase activity reaches the maximum (lanes 5 and IO). Tec can associate with Shc in vivo. We then investigated the role of Tec in vivo. The Shc protein is an adapter peptide containing an SH2 motif, and overexpression of Shc can transform NIH 3T3 fibroblasts.2sShc is known to transduce mitogenic signals from PTKs to the Ras pathway through the association with Grb-2 and SOSproteins.2hBecause Shc is abundantly expressed in hematopoietic tissues, we examined whether Tec is involved in the Shc-mediated signalings. Anti-Shc immunoprecipitates from BaF3 cells with or without the IL-3 stimulation were blotted with anti-Tec serum. As shown in Fig 7, Tec was shown to be associated with Shc protein irrespective of IL-3 stimulation. Therefore, Tec is capable of making a stable complex with Shc in vivo. DISCUSSION We have characterized the Tec protein-tyrosine kinase in hematopoietic cell lines, showing the predominant structure of the Tec protein and its involvement in the IL-3 signaling pathway. We show here that type 111 and type IV of Tec are the predominant forms in mouse tissues. Although these two types of Tec could be indeedidentified in several mouse organs, only type IV could be observed in all hematopoietic cell lines examined (Sato et al, personal communication, July 1994). The difference of biologic roles between type 111 and IV is currently under investigation. There have been several reports of alternatively spliced messages for cytoplasmic PTKs. Neuronal c-Src has a six-amino acid insertion in its noncatalytic domain2’ anda totally different open reading frame can be usedfroman alternatively spliced c-src message in chicken skeletal muscle.2RAmino acid insertions are also reported in the Lyn protein.29The N-terminal alteration of PTKs is known in the case of c-Abl.’ There are four different c-Ab1 mRNAs with divergent 5”termini; only one of them (type IV) can encode a peptide with a myristylation signal. Thus, alternative splicing mechanismcanregulate subcellular localization of PTKs.In our present report, neither form of Tec type 111 or type IV has putative myristylation sites or C-terminal tyrosine residues corresponding to Tyr-527 in c-Src. The biologic significance of the two predominant forms of 5”region in the tec message is yet to be shown. Both types of cDNAs encode the same N-terminus of Tec, but they differ in the 5”noncoding region. Marth et a!’’ reported that deletion of 5”noncoding sequence of the Ick message, including 3 ATG sequences, significantly increased translational efficiency of ~ 5 6 They . ~found ~ that morethan 65% of mammalian genes withsuch5’-ATG sequences are proto-oncogenes. Messages of fgr, fin, hck, Ick, and src actually possess the multiple 5’-ATG sequences that are not used for the translational start sites. Thus, they hypothesized that presence of multiple ATG sequences in the 5”noncoding region of PTK messages suppresses their translational efficiency in vivo and prevents malignant transformation. Interestingly, the IA sequence in the tec message From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 349 TECIS INVOLVED IN IL-3SIGNALINGPATHWAY IL-3 4- Tec 4- IgH in the system of myeloid cells. Although IL-3or granulocytemacrophage colony-stimulating factor can induce tyrosine phosphorylation in a set of cellular proteins in myeloid cells, it was unclear what kinases mediate such signals in vivo. Recently, Jak-2 has been shown to be activated in response to erythropoietin," IL-3;' and IL-6..'*However, significance of Jak-2 in the mitogenic signaling pathways of these cytokines is still obscure. Because Tec is expressed no less than known cytoplasmic PTKs in myeloidcells,13 it wouldbe reasonable to presume that the Tec kinase plays an important role in vivo. We could actually show that Tec is involved in the stem cell factork-Kit signaling m e c h a n i ~ m .We ~~ could also show that the N-terminal unique domain of Tec is involved in IL-3 signaling and is associated with the Lyn kinase in myeloidcells.3" In this report, we haveshown that IL-3 stimulation can induce tyrosine phosphorylation of pp7OTC'in myeloid cells as well as in pro-B cells. Furthermore, Tec was shown to associate with Shc in vivo. Although our data do not fully clarify the function of Tec, they support the idea thatTec is involvedin the mitogenic signaling mechanism of IL-3. Tec proteins are composed of four domains, namely, an N-terminal unique domain, an SH3 domain, an SH2 domain, and a kinase domain. The overall structure of Tec is, therefore, similar to that of the Src family members. In contrast, Jak kinases have rather unusual structures. They are composed of an N-terminal unique domain, a kinase-like domain, and a kinase domain. They do not contain any SH2 or SH3 motifs. Considering the difference between the structures of Tec and Jakkinases, it is likely that theyare assigned divergent roles in vivo. Tang et a1'9 could indeed prove that Tec is involved in the stem cell factodc-Kit signaling system, while Jak-2 is not. Because IL-3 can exert pleiotropic effects on hematopoietic cells, Tec and Jak-2 may play differential roles in the IL-3 signaling pathways. ACKNOWLEDGMENT Fig 7. Tec can associate with Shc. The Shc protein was immunoprecipitated from BaF3 cells with (+) or without (-1 11-3 stimulation and was blotted with anti-Tec serum. The positions of Tec and lg heavy chain (IgH) are indicated by arrows. MurineIL-3 was kindlyprovided (Tokyo, Japan). by KirinBrewery CO Ltd REFERENCES 1. BolenJB: contains 4 ATG sequences that are not used for the translational start sites of the Tec protein. Therefore, the rec message without the IA sequence may be translated much more efficiently than that with the I A sequence. We are currently investigating whether the presence of the IA sequence in the message may affect the Tec activity in vivo. Accumulating evidence has proved that several PTKs of the Src family are involved in the mitogenic mechanism of lymphocytes. Lck, for example, is associated withand activated by antibody-stimulated CD4/CD8 molecules in T cells.7' Hatakeyama et alz2could demonstrate the physical association of the Lck kinase and the IL-2 receptor. The Fyn kinase has been revealed to belinked to T-cell receptor complex.33The Lyn kinase has been also shown to be activated by the stimulation of surface IgM molecules in B cells.34Importantly, some of them are already known to play a role in the process of malignant transformation.3s However, little was understood about such "key kinases" Nonreceptor tyrosine protein kinases. Oncogene 8:202S,1993 2. 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For personal use only. 1995 85: 343-350 Tec protein-tyrosine kinase is involved in interleukin-3 signaling pathway H Mano, Y Yamashita, K Sato, Y Yazaki and H Hirai Updated information and services can be found at: http://www.bloodjournal.org/content/85/2/343.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.