Myristoylation is a co-translational maturation process of proteins. It is extremely specific for the cosubstrate (myristoyl-CoA) and for the substrate protein that should bear a glycine at the Nterminus of the protein to be myristoylated. This acylation is catalyzed by the myristoyl-CoA :protein N-myristoyltransferase. Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae. Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues. In earlier studies, we demonstrated the existence of a microsomal N-myristoyltransferase from the murine leukemia cell line L1210 [Boutin, J. A., Clarenc, J.-P., Ferry, G., Ernould, A. P., Remond, G., Vincent, M. & Atassi, G. (1991) E m J. Biochem. 201,257-2631, a feature which is not shared by yeast, and examined the N-myristoyltransferase activities associated with L1210 cytosol. In the present work, we purified to homogeneity one of the isoforms (A) of the transferase from L1210 cytosol. The purified enzyme showed on SDSPAGE an apparent molecular mass of 67.5 kDa, distinct from the 53-kDa yeast cytosolic enzyme. The purified enzyme from L1210 cytosol could be labeled with ['4C]myristoyl-CoA. Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source. A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH,-terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins. We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme. For example, a myristoyltetrahydroquinolein derivative showed an IC,, of about 0.1 pM. Based on both biophysical and biochemical evidence, the N-myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme.Myristoyl-CoA :protein N-myristoyltransferase (NMT) is the enzyme responsible for a co-translational modification involving the rare fatty acid, myristic acid, and having as a binding site a glycine residue at the N-terminal of target proteins [l]. Numerous oncogene products from mammalian transforming retroviruses are either myristoylated or expressed as gag-onc fusion proteins which are myristoylated on the N-terminal glycine of the gag protein [2]. This maturation process has been proved to be essential in several types of pathology either by addressing the implicated oncogene product (like src) to a specific site at the plasma membrane level [3-51 or by permitting the regular assembly of the retroviral structural protein gag or the equivalent in other types of viruses [6-91. In both cases, using molecular biology tools, it has been demonstrated that this maturation...

The activity catalyzed by N-myristoyl tranferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accomodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively).As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificy.Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity .A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (I(&) of 0.2 mM.Myristoylation is a post-translational acylation of proteins catalyzed by NMT [l, 21. From the literature, a growing number of proteins undergo this modification, provided that as an absolute requirement, these proteins bear a glycine at their Nterminus (see [3, 41 for reviews). Amongst the proteins so acylated, three categories can be defined : endogenous proteins (such as protein kinase A, guanyl-nucleotide-binding protein, NADHlcytochrome-b, reductase); oncogene products like pp60v-src, p561ck, p59fyn or p62yes; virus structural proteins such as gag from various types of retrovirus, for example Moloney murine leukemia virus and human immunodefficient virus (see [5, 61 for reviews).Several lines of evidence showed both in virology and cancerology that the suppression by directed mutagenesis of the site of myristoylation leads to (a) the suppression of the transforming potency of pp6Ov-src [7 -91; (b) the assembly of non-infectious viral particles for VPO [lo, 111 and VP1 [12], and (c) the inhibition of viral-particle formation and budding for gag [13-151. Furthermore, numerous viral oncogenes cfes, abl, ras) are expressed in cancerous cell lines as gag-onc fusion proteins which are the active transforming species of the corresponding

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The major tyrosine protein kinase, HPK40, isolated from HL‐60, the preparation of which is described elsewhere (Ernould, A.P., Ferry, G., Barret, J.M., Genton, A. and Boutin, J.A., Eur. J. Biochem., 214, 503–514), was investigated as to its specificity on a number of peptides and proteins. It was found that HPK40 can phosphorylate histones (except histone H4), casein, acid‐treated enolase, actin and tubulin but not calmodulin. Phosphorylation specificity of HPK40 was investigated using over a hundred peptidic structures. HPK40 is not related to the ‘src’ family and does not phosphorylate efficiently either the tetrapeptide NEYT derived from the pp60src autophosphorylation domain or the corresponding peptide RRsrc, RRLIEDNEYTARG. VALYDYESR from the SH3 domain of pp60c‐src is recognized as a substrate with a high phosphorylation level. DEDYIQD, derived from the phosvitin/casein kinase II, was also highly phosphorylated. In order to determine the minimal recognition sequence of HPK40, the phosphorylation of about 60 dito tetrapeptides was investigated. Some of the tetrapeptides, such as *EEYE and NEYE, were well phosphorylated. Even some tripeptides, such as EYE, DYM, TYS and KYE, were recognized by HPK40, while none of the tested dipeptides was recognized as substrate. Sequences of peptides from DRVYHPF (angiotensin), LEEEEEAYGWMDF (minigastrin) and QEEYSAM (from H‐rasl) were examined as substrates. The presence of one or several acidic residues on the Nα‐side of tyrosine residue was identified as the only apparently favorable determinant. These results are steps towards the minimum recognition sequence, which in turn will serve as a lead for chemical modifications in view of obtaining a specific, low‐molecular‐weight, inhibitor of this human tyrosine protein kinase.

The major tyrosine protein kinase from HL60 (a human non-differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromatographic steps : DEAE-Sepharose, casein-agarose, cibacron-blue -agarose and hexyl-agarose. The purification resulted in more than 1000-fold enrichment in angiotensin I1 phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP [ys] and autophosphorylation of the enzyme in the presence of [Y-~~PIATP, all led to the identification of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl andfes oncogene products. Phosphorylation of angiotensin 11 and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The K, for ATP was 2 pM and the V,, 3.1 nmol min-' . mg-' using angiotensin fI as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40-kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.Protein phosphorylation plays a key role in the control of cellular communication [l]. Amongst the enzymes catalyzing the phosphorylation of other proteins (on specific amino acid residues), the tyrosine protein kinases (TPK) are known to be involved in the regulation of growth signal transmission and cellular transformation [2], according to their functional similarity to growth factor receptors and oncogene products [3]. A growing number of TPK(s) have been identified, mostly from molecular cloning of protein kinase genes rather than through enzyme purification [4]. TPK(s) can be classified in three categories [5][6][7]: (a) the transmembrane-receptor-associated TPK activity as found, for instance, in epidermal growth factor EGF, platelet growth factor PDGF and insulin receptors; (b) the TPK(s) linked to the inner face of the plasma membrane represented by the src family with src, yes, f g r , f m , lyn, hck, lck and blk oncogene products which are all myristoylated [8] and (c) the cytosolic TPK(s) with major representatives being abl and fes/fgr oncogene products.From a molecular pharmacological point of view, TPK(s) are new targets for inhibitors [9] To understand the cellular function of these TPKs one needs to characterize them on the basis of classical biochemistry studies (purification, molecular identification, kinetic data, effect of modulators, etc.). A major example was recently provided by Litwin et al. [24] and Cheng et al. [25], on a TPK specifically phosphorylating the p34cdc2 kinase ...