Modified protein derived from protein kinase N

Details Modified protein derived from protein kinase N Modified protein derived from protein kinase N

1996-07-24

2003-12-09

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000, C514S002600, C514S012200, C435S194000, C435S320100, C435S252300, C435S252330, C435S325000, C536S023100, C536S023200, C536S023500

Reexamination Certificate

active

06660837

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a modified amino acid sequence of Protein Kinase N, and more particularly to a modified amino acid sequence of PKN having activated Rho protein binding activity.
BACKGROUND OF THE INVENTION
A group of low-molecular-weight GTP-binding proteins (G-proteins) with molecular weights of 20,000-30,000 with no subunit structures are observed in organisms. To date, over fifty or more members have been found as the super family of the low-molecular-weight G-proteins in a variety of organisms, from yeast to mammals. The low-molecular-weight G-proteins are divided into four families of Ras, Rho, Rab and the others based on homologies of amino acid sequences. It has been revealed that the small G-proteins control a variety of cellular functions. For example, the Ras protein is considered to control cell proliferation and differentiation, and the Rho protein is considered to control cell morphological change, adhesion and motility.
The Rho protein, having GDP/GTP-binding activity and intrinsic GTPase activity, is believed to be involved in cytoskeletal rearrangement in response to extracellular signals such as lysophosphatidic acid (LPA) and certain growth factors. When the inactive GDP-binding Rho is stimulated, it is converted to the active GTP-binding Rho protein (hereinafter referred to as “the activated Rho protein”) by GDP/GTP exchange proteins such as Smg GDS, Dbl or Ost. The activated Rho protein then acts on target proteins to form stress fibers and focal contacts, thus inducing the cell adhesion and motility (Experimental Medicine, Vol. 12, No. 8, 97-102 (1994); Takai, Y. et al., Trends Biochem. Sci., 20, 227-231 (1995)). On the other hand, the intrinsic GTPase activity of the Rho protein converts the activated Rho protein to the GDP-binding Rho protein. This intrinsic GTPase activity is activated by what is called GTPase-activating proteins (GAP) (Lamarche, N. & Hall, A. et al., TIG, 10, 436-440 (1994)).
The Rho family proteins, including RhoA, RhoB, RhoC, Rac1, Rac2 and Cdc42, share more than 50% sequence identity with each other. The Rho family proteins are believed to be involved in inducing the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors (A. J. Ridley & A. Hall, Cell, 70, 389-399 (1992); A. J. Ridley & A. Hall, EMBO J., 13, 2600-2610 (1994)). The subfamily Rho is also considered to be implicated in physiological functions associated with cytoskeletal rearrangements, such as cell morphological change (H. F. Parterson et al., J. Cell Biol., 111, 1001-1007 (1990)), cell adhesion (Morii, N. et al., J. Biol. Chem., 267, 20921-20926 (1992); T. Tominaga et al., J. Cell Biol., 120, 1529-1537 (1993); Nusrat, A. et al., Proc. Natl. Acad. Sci. USA, 92, 10629-10633 (1995)*; Landanna, C. et al., Science, 271, 981-983 (1996)*, cell motility (K. Takaishi et al., Oncogene, 9, 273-279 (1994), and cytokinesis (K. Kishi et al., J. Cell Biol., 120, 1187-1195 (1993); I. Mabuchi et al., Zygote, 1, 325-331 (1993)). (An asterisk hereinafter indicates a publication issued after the first filed application which provides the right of the priority of the present application.) In addition, it has been suggested that the Rho is involved in the regulation of smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); M. Noda et al., FEBS Lett., 367, 246-250 (1995); M. Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996)*; K. Kimura, et al., Science, 273, 245-248(1996)*), and the expression of phosphatidylinositol 3-kinase (PI3 kinase) (J. Zhang et al., J. Biol. Chem., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI 4,5-kinase) (L. D. Chong et al., Cell, 79, 507-513 (1994)) and c-fos (C. S. Hill et al., Cell, 81, 1159-1170 (1995)).
Recently, it has also be found that Ras-dependent tumorigenesis is suppressed when the Rho protein of which the amino acid sequence has been partly substituted is introduced to cells, revealing that the Rho protein plays an important role in Ras-induced transformation, that is, tumorigenesis (G. C. Prendergast et al., Oncogene, 10, 2289-2296 (1995); Khosravi-Far, R. et al., Mol. Cell. Biol., 15, 6443-6453 (1995)*; R. Qiu et al., Proc. Natl. Acad. Sci. USA, 92, 11781-11785 (1995)*; Lebowitz, P. et al., Mol. Cell, Biol., 15, 6613-6622 (1995)*).
It has also been demonstrated that mutation of GDP/GTP-exchange proteins which act on the Rho protein results in cell transformation (Collard, J., Int. J. Oncol., 8, 131-138 (1996)*; Hart, M. et al., J. Biol. Chem., 269, 62-65 (1994); Horii, Y. et al., EMBO J., 13, 4776-4786 (1994)).
In addition, the Rho protein has been elucidated to be involved in cancer cell invasion, that is, metastasis (Yoshioka, K. et al., FEBS Lett., 372, 25-28 (1995)). The cancer cell invasion is closely associated with changes in cancer cell activity to form cell adhesion. In this context, the Rho protein is also known to be involved in the formation of cell adhesion (see above Morii, N. et al. (1992); Tominaga, T. et al. (1993); Nusrat, A. et al. (1995); Landanna C. et al. (1996)*).
On the other hand, a novel protein kinase having a molecular weight of approximately 120 kDa (hereinafter referred to as PKN or Protein Kinase N) has recently been isolated, and the whole amino acid sequence thereof has been determined. Furthermore, PKN has been proved to have a catalytic region highly homologous to that of Protein Kinase C and actually has serine/threonine kinase activity (Mukai, H. & Ono, Y. Biochem. Biophys. Res. Commun. 199, 897-904 (1994), Mukai, H. et al., Biochem. Biophys. Res. Commun. 204, 348-356 (1994), and Mukai, H. et al., Biochem. Biophys. Acta 1261, 296-300 (1995)). Substitution of Arg for Lys at position 644 of PKN leads to loss of the protein kinase activity (Mukai, H. et al., ibid.).
The protein kinase activity is activated by unsaturated fatty acids such as arachidonic acid (Mukai, H. et al., Biochem. Biophys. Res. Commun., 204, 348-356 (1994); and Kitagawa, M. et al., Biochem. J., 310, 657-664 (1994)). cDNAs of PKN of human beings, rat, and Xenopus have been cloned, and the amino acid sequences thereof have been determined (Mukai, H. & Ono, Y., Biochem. Biophys. Res. Commun., 199, 897-904 (1994); Mukai, H. et al., Biochim. Biophys. Acta., 1261, 296-300 (1995)). PKN from human is a protein of 942 amino acid residues, and the amino acid sequence of the carboxyl-terminal catalytic region is highly homologous to the amino acid sequence of the catalytic region of Protein Kinase C. Therefore, PKN is often called Protein Kinase C-related kinase 1 (Palmer, R. H. & Parker, P. J., FEBS Lett., 356, 5-8 (1994)).
The amino-terminal regulatory region of PKN contains some of leucine zipper sequences, and a polybasic region is located immediately at the amino terminal side of the leucine zipper sequence. Furthermore, it has been reported that, there are at least two isozymes concerning PKN (protein kinase C-associated kinases 2 and 3) (Palmer, R. H. & Parker, P. J., FEBS Lett. 356, 5-8 (1994)).
It is only recently (after the first filed application which provides the right of the priority of the present application) that a several proteins have been identified as candidates of Rho-targets in mammals different from PKN: citron (Madaule, P. et al., FEBS Lett., 377, 243-248 (1995)*), rhophilin (Watanabe, G. et al., Science, 271, 645-648 (1996)*), p160
ROCK
(Ishizaki, T. et al., EMBO J. 15, 1885-1893(1996)*), Rho-associated kinase (Matsui, T. et al., EMBO J., 15, 1885-1893 (1996)*), ROK&agr;(Leung, T. et al., J. Biol. Chem., 270, 29051-29054 (1995)*), rhotekin (Reid, T. et al., J. Biol. Chem., 271, 9816-9822 (1996)*), and myosin binding subunit(K. Kimura, et al., Science 273, 245-248(1996)*). In addtion, Protein Kinase C1 (PKC1) (Nonaka, H. et al., EMBO J. 14, 5931-5938(1995)*) and 1,3-&bgr;-glucan synthase (Drgonova, J. et al., Science 272, 277-279(1996)*; Qadota, H. et al., Science 272, 279-281(1996*) have been identified as candidates of Rho-targets in yeasts (S

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