Differential Expression of Low Mr GTP-binding Proteins in Human Megakaryoblastic Leukemia Cell Line, MEG-01, and their Possible Involvement in the Differentiation Process

Summary

The expression of various low Mr GTP-binding proteins at various states of differentiation of a human megakaryoblastic leukemia cell line, MEG-01, was analyzed using thermocycle amplification of mRNA and immunoblotting. MEG-01 cells were found to express mRNAs of rap1A, rap1B, rap2B, ralA, rhoA, rac1, rac2, CDC42Hs, rab1, rab3B, rab6, ram and ran, but not rab4, and the proteins of Rap 1, Rap2, RhoA, Rac1, Rac2, Rab3B, Rab4, Rab6 and Rab8 were expressed. Differentiation of MEG-01 cells induced by 100 nM 12-O-tetradecanoylphorbol-13-acetate revealed the considerable increases in mRNA expression of rap1B, rab3B, rabA, ram and ran whereas the levels of rap2B, rhoA and rac1 decreased. During the differentiation process, significant changes in protein levels of Rap1, RhoA, Rac1, Rac2, Rab3B, Rab4 and Rab6 were observed among three subcellular (cytosol, Triton X-100-soluble membrane and -insoluble cytoskeleton) fractions. The present investigation may be useful for the study of the megakaryocyte differentiation.

• References

• 1 Valencia A, Chardin P, Wittinghofer A, Sander C. The ras protein family:evolutionary tree and role of conserved amino acids. Biochemistry 1991; 30: 4637-4648
• 2 Takai Y, Kaibuchi K, Kikuchi A, Kawata M. Small GTP-binding proteins. Int Rev Cytol 1992; 133: 187-230
• 3 Barbacid M. ras Genes. Ann Rev Biochem 1987; 56: 779-827
• 4 Kaziro Y, Itoh H, Kozasa T, Nakafuku M, Satoh T. Structure and function of signal-transducing GTP-binding proteins. Ann Rev Biochem 1991; 60: 349-400
• 5 Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, Saito H. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 1985; 66: 1384-1392
• 6 Ogura M, Morishima Y, Okumura M, Hotta T, Takamoto S, Ohno R, Hirabayashi N, Nagura H, Saito H. Functional and morphological differentiation induction of a human megakaryoblastic leukemia cell line (MEG-01 s) by phorbol diesters. Blood 1988; 72: 49-60
• 7 Horvath AR, Muszbek L, Kellie S. Translocation of pp60^C-SRCto the cytoskeleton during platelet aggregation. EMBO J 1992; 11: 855-864
• 8 Nagata K, Itoh H, Katada T, Takenaka K, Ui M, Kaziro Y, Nozawa Y. Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol:one is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein. J Biol Chem 1989; 264: 17000-17005
• 9 Nagata K, Nozawa Y. Subcellular localization of a low Mr GTP-binding protein, c25KG, in resting and stimulated human platelets. Biochem Biophys Res Commun 1993; 195: 1081-1088
• 10 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1988; 162: 156-159
• 11 Gyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 1988; 85: 7652-7656
• 12 Laemmli UK. leavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 13 Nagata K, Hattori T, Hachiya T, Takahashi T, Nozawa Y. Purification and characterization of a low Mr GTP-binding protein, c25KG, from human platelet membranes. Biochem Biophys Acta 1992; 1160: 193-198
• 14 Bradford MM. A rapid and simple method for the quantitation of quantities of protein utilizing the principle of protein-dye. Analyt Biochem 1976; 72: 248-254
• 15 Nagata K, Nozawa Y. Purification and characterization of two GTP-binding proteins of 22 kDa from human platelet membranes. FEBS Let 1988; 237: 113-117
• 16 Ohmori T, Kikuchi A, Yamamoto K, Kim S, Takai Y. Small molecular weight GTP-binding proteins in human platelet membranes:Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000. J Biol Chem 1989; 246: 1877-1881
• 17 Nagata K, Satoh T, Itoh H, Kozasa T, Okano Y, Doi T, Kaziro Y, Nozawa Y. The ram:a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library. FEBS Lett 1990; 275: 29-32
• 18 Farrell FX, Ohmstede CA, Reep BR, Lapetina EG. cDNA sequence of a new ras-related gene (rap 2b) isolated from human platelets with sequence homology torapl . Nucleic Acids Res 1990; 18: 4281
• 19 Karniguian A, Zahraoui A, Tavitian A. Identification of small GTP-binding proteins in human platelets:Thrombin-induced phosphorylation ofrab3B,rab6, andrab8 proteins. Proc Natl Acad Sci USA 1993; 90: 7647-7651
• 20 Nemoto Y, Namb T, Teru-uchi T, Ushikubi F, Morii N, Narumiya S. A rho gene product in human platelets:I Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein. J Biol Chem 1992; 267: 20916-20920
• 21 Narumiya S, Morii N. rho gene products, botulinom C3 exoenzyme and cell adhesion. Cell Signal 1993; 05: 09-19
• 22 Morii N, Teru-uchi T, Tominaga T, Kumagai N, Kozaki S, Ushikubi F, Narumiya S. A rho gene product in human platelets:II effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. J Biol Chem 1992; 267: 20921-20926
• 23 Aepfelbacher M, Vauti F, Weber PC, Glomset JA. Spreading of differentiating human monocytes is associated with a major increase in membranebound CDC42. Proc Natl Acad Sci USA 1994; 91: 4263-4267
• 24 Van der MeulenJ, hullar BRP, Chancellor-Maddison KA. Association of a 24-KDa GTP-binding protein, Gn24, with human platelet α-granule membranes. FEBS Lett 1991; 291: 122-126
• 25 Witte DP, Harris RE, Jenski LJ, Lampkin BC. Megakaryoblastic leukemia in an infant:establishment of a megakaryocytic tumor cell line in athymic nude mice. Cancer 1986; 58: 238-244
• 26 Adachi M, Ryo R, Yoshida A, Sugano W, Yasunaga M, Saigo K, Yamaguchi N, Sato T, Sano K, Kaibuchi K, Takai T. Induction of smg p21/rap1A/Krev-1 p21 gene expression during phorbol ester-induced differentiation of a human megakaryocytic leukemia cell line. Oncogene 1992; 7: 323-329
• 27 Pizon V, Lerosey I, Chardin P, Tavitian A. Nucleotide sequence of a human cDNA encoding a ras-related protein (rapIB). Nucleic Acid Res 1988; 16: 7719
• 28 Nagata K, Nagao S, Nozawa Y. Low Mr GTP-binding proteins in human platelets;cyclic AMP-dependent protein kinase phosphorylates m22KG(I) in membrane but not c21KG in cytosol. Biochem Biophys Res Commun 1989; 160: 235-242
• 29 Kawata M, Kikuchi A, Hoshijima M, Yamamoto K, Hashimoto E, Yamamura H, Takai Y. Phosphorylation of smg p21, a ras-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin El in intact human platelets. H Biol Chem 1989; 264: 15688-15695
• 30 Fischer TH, Gatling MC, McCormick F, Duffy CM, White IIGC. Incorporation of RaplB into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium. J Biol Chem 1994; 269: 17257-17261
• 31 Bokoch GM. Biology of the Rap proteins, members of the ras superfamily of GTP-binding proteins. Biochem J 1993; 289: 17-24
• 32 Madaule P, Axel R. A novel ras-related gene family. Cell 1985; 41: 31-40
• 33 Didsbury J, Weber RF, Bokoch GM, Evans T, Snyderman R. rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem 1989; 264: 16378-16382
• 34 Shinjo K, Koland JG, Hart MJ, Narasimhan V, Johnson DJ, Evans T, Cerione R. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K):identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein C DC42. Proc Natl Acad Sci USA 1990; 87: 9853-9857
• 35 Munemitsu S, Innis MA, Clark R, McCormick F, Ullrich A, Polakis P. Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene C DC42. Mol Cell Biol 1990; 10: 5977-5982
• 36 Vincent S, Jeanteur P, Fort P. Growth-regulated expression of rhoG, a new member of the ras homolog gene family. Mol Cell Biol 1992; 12: 3138-3148
• 37 Drivas GT, Shih A, Coutavas E, Rush MG, D’Eustachio P. Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line. Mol Cell Biol 1990; 10: 1793-1798
• 38 Paterson HF, Self AJ, Garrett MD, Just I, Aktories K, Hall A. Microinjection of p21 rho induces rapid changes in cell morphology. J Cell Biol 1990; 111: 1001-1007
• 39 Ridley AJ, Paterson HF, Johnson CL, Diekman nD, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992; 70: 401-410
• 40 Knaus UG, Heyworth PG, Evans T, Camutte JT, Bokoch GM. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac2. Science 1991; 254: 1512-1515
• 41 Diekmann D, Abo A, Johnston C, Segal AW, Hall A. Interaction of Rac with p67 phox and regulation of phagocytic NADPH oxidase activity. Science 1994; 265: 531-533
• 42 Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992; 70: 389-399
• 43 Nagata K, Suzuki T, Shibagaki Y, Mizumoto K, Okano Y, Kaziro Y, Nozawa Y. Characterization and site-directed mutagenesis of a low Mr GTP-binding protein., ram p25, expressed in Escherichia coli. J Biol Chem 1992; 267: 16000-16006
• 44 Nagata K, Okano Y, Suzuki T, Nozawa Y. Evidence for the presence of a low Mr GTP-binding protein, ram p25, in human platelet membranes. Platelets 1993; 04: 268-274
• 45 Nuoffier C, Balch WE. GTPases:multi-functional molecular switches regulating vesicular traffic. Annu Rev Biochem 1994; 63: 949-990
• 46 Matsumoto T, Beach D. Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase. Cell 1991; 66: 347-360
• 47 Nagata K, Takemasa T, Alam S, Hattori T, Watanabe Y, Nozawa Y. Cloning of cDNAs encoding a cell-cycle-regulatory GTP-binding low-Mr (GBLM) protein, Ran/TC4, from micronucleated Tetrahymena thermophila and amicronucleated Tetrahymena pyriformis. Gene 1994; 144: 123-125
• 48 Mindong R, Drivas G, D’Eustachio P, Rush MH. Ran/TC4:a small GTP-binding protein that regulates DNA synthesis. J Cell Biol 1993; 120: 313-323
• 49 Moore MS, Blobel G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 1993; 365: 661-664