Peptide substrates phosphorylated by P21-activated protein...

Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues

Reexamination Certificate

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C435S023000, C435S226000, C435S320100, C435S069100, C530S328000, C530S329000, C530S350000, C530S380000

Reexamination Certificate

active

06228989

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention involves peptides and their use in assays for protein kinase activity.
BACKGROUND OF THE INVENTION
&ggr;-PAK (also known as PAK2), a member of the p2I-activated protein kinase (PAK) family [Lim, L., et al.,
Eur. J. Biochem
., 242:171-185 (1996); Sells, M. A.,
Trends Cell Biol
., 7: 180-167 (1997); and Jakobi, R., et al.,
J. Biol. Chem
.,271:6206-6211 (1996) ] and formerly designated PAK I, is a serine/threonine kinase of 58 000 Da found in a number of tissues and species [Tahara, S. M., et al.,
J. Biol. Chem
., 256:11558-11564 (1981); Tahara, S. M., et al.,
Eur. J. Biochem
., 126:395-399 (1982); Tuazon, P. T., et al.,
Eur. J. Biochem
., 129:205-209 (1982); Tuazon, P. T., et al.,
J. Biol. Chem
., 259,541-546 (1984); Rooney, R. D., et al.,
FASEB J
., 6:1852 (Abstract) (1992); Rooney, R. D., et al., J.
Biol. Chem
., 271:21498-21504 (1996) and Benner, G. E., et al.,
J. Biol. Chem
., 270:21121-21128 (1995)]. &ggr;-PAK was first identified in rabbit reticulocytes as an inactive holoenzyme that could be activated in vitro by limited proteolysis with trypsin, chymotrypsin, or a Ca
2+
-stimulated protease, hence the initial nomenclature of protease-activated kinase (PAK) I [Tahara, S. M., et al., (1981), supra and Tahara, S. M., et al., (1982), supra]. Limited proteolysis of the inactive holoenzyme with trypsin produces a catalytically active peptide of 37 000 Da that contains the catalytic domain and a part of the regulatory domain [Jakobi, R., et al., supra]. The enzyme from rabbit reticulocytes has been shown to be highly homologous to &ggr;-PAK from human [Manser, E., et al.,
Nature
, 367:40-46 (1994)] and rat [Martin, G. A., et al.,
EMBO J
., 14:1970-1978 (1995) with some homology to STE 20 from yeast [Ramer, S. W., et al.,
Proc. Natl. Acad. Sci. U.S.A
., 90:452-456 (1993)]. Like other PAK enzymes [Lim, L., et al., supra and Sells, M. A., supra], &ggr;-PAK can bind small G proteins such as Cdc42 and Rac in the presence of GTP to stimulate autophosphorylation, resulting in activation of the protein kinase activity.
&ggr;-PAK is also proteolytically activated in vivo during early apoptosis, and in vitro, by Caspase 3 (CCPP32) [Rudel, T., et al.,
Science
, 276:1571-1574 (1997); Lee, N., et al.,
Proc. Natl. Acad, Sci U.S.A
., 94:13642-13647 (1997); and Walter, B. N., et al., J.
Biol. Chem
., 273:28733-28739 (1998)]. Cleavage produces a regulatory domain of 27,000 Da and an active catalytic domain of 34,000 Da [Walter, B. N., et al., supra].
&ggr;-PAK activity is elevated in serum-starved and quiescent cells and is drastically reduced in actively dividing cells. In frog eggs, &ggr;-PAK activity and protein are high in frog oocytes and are reduced following fertilization and at the 2-cell stage. In the 4- and 16-32-cell stages, &ggr;-PAK reappears, but mainly as the inactive form [Rooney, R. D., et al., (1996), supra]. Injection of &ggr;-PAK into one blastomere of 2-cell frog embryos results in cleavage arrest, while the noninjected blastomere continues to divide through mid- to late-cleavage. These observations suggest that &ggr;-PAK is involved in the maintenance of cells in a nondividing state [Rooney, R. D., et al, (1996), supra].
&ggr;-PAK has been shown to phosphorylate a number of protein substrates such as histones 2B and 4 [Tahara, S. M., et al., (1981), supra]; myosin light chain from smooth and skeletal muscle [Tuazon, P. T., et al., (1982), supra and Tuazon, P. T., et al, (1984), supra]; translation initiation factors elF-3, elF-4B, and eIF-4F [Tahara, S. M., et al., (1982), supra and Tuazon, P. T., et al.,
J. Biol Chem
., 264:2773-2777 (1989)]; and avian and Rous sarcoma virus nuclear capsid protein NC [Leis, J., et al.,
J. Biol. Chem
., 259:7726-7732 (1984); Fu, X., et al.,
J. Biol. Chem
., 260:99411-9947) (1985); and Fu, X., et al.,
J. Biol. Chem
., 263:2134-2139 (1988)]. Phosphorylation of myosin light chain in smooth muscle by &ggr;-PAK increases the actin-activated myosin ATPase activity to the same extent as that observed upon phosphorylation by the Ca
2+
calmodulin-dependent myosin light chain kinase [Tuazon, P. T., et al., (1984), supra]. In the Rous sarcoma virus nucleocapsid protein NC, phosphorylation by &ggr;-PAK at serine 40 increases the affinity for single-strand RNA by up to 100-fold [Fu, X., et al., (1985, supra]. Studies with site-specific mutants of NC indicate phosphorylation by &ggr;-PAK can regulate binding to viral RNA [Fu, X., et al., (1988), supra]. Thus, it appears from the diversity of substrates that &ggr;-PAK may be involved in regulation of multiple pathways of cell metabolism.
One approach to elucidate the role of protein kinases in general, and &ggr;-PAK in particular, is to identify their substrates. The search for possible substrates can be facilitated by a knowledge of the amino acids critical for efficient phosphorylation. It is generally accepted that the primary sequence around the phosphorylation site plays a crucial role in the recognition of substrates for a number of protein kinases [Kennelly, P. J., et al.,
J. Biol. Chem
., 266:15555-15558 (1991)]. In these studies, determinants for phosphorylation by &ggr;-PAK have been characterized using synthetic peptides patterned after the phosphorylation site identified as Ser 40 in the sequence PKKRKSGL in the Rous Sarcoma virus nuclear capsid protein NC, which is similar to &ggr;-PAK sites identified in other proteins [Leis, J., et al., supra]. A model peptide KKRKSAA was synthesized and the amino acids around the phosphorylation site in the model peptide were systematically substituted with other amino acid residues to determine the minimum phosphorylation sequence for &ggr;-PAK. The rates of phosphorylation of the peptides by &ggr;-PAK were compared with those obtained with two other protein kinases that require basic residues around the phosphorylation site, protein kinase A (PKA) and protein kinase (PKC). On the basis of the substrate specificity requirements, a synthetic peptide that could be useful in characterizing &ggr;-PAK in complex mixtures of protein kinases, such as crude extracts of cells or tissues, has been identified.
SUMMARY OF THE INVENTION
The present invention satisfies the need for a substrate that can be used to detect and differentiate &ggr;-PAK from other protein kinases present in a sample. A peptide that is a selective substrate for PAK is provided, having the amino acid sequence:
X
1
X
2
X
3
R
4
X
5
X
6
X
7
X
8
  (1)
wherein X
1
and X
8
together equal 0 to 4 amino acids, each of which is independently selected; X
2
, X
5
or both X
2
and X
5
is an acidic amino acid; X
3
is arginine or lysine; R
4
is arginine; X
5
and X
7
are not proline; and X
6
is serine or threonine. Preferably, at least one of X
2
and X
5
is selected from the group consisting of aspartic acid, glutamic acid, phosphoserine, and phosphothreonine. The most preferred peptide substrates which are highly specific substrates for PAK are AKRESAA (SEQ ID NO:1) and EKRASAA (SEQ ID NO:2).
Another embodiment of the present invention is a peptide that is a highly efficient substrate for a protein kinase, such as PAK, which has the amino acid sequence:
X
1
X
2
X
3
R
4
X
5
X
6
X
7
X
8
  (2)
wherein X
1
, X
2
, X
7
, and X
8
together equal 1 to 6 amino acids, each of which is independently selected; at least one of X
2
and X
5
is an acidic or basic amino acid; X
3
is arginine or lysine; R
4
is arginine; X
5
is an independently selected amino acid; and X
6
is serine or threonine.
The peptide substrates of the present invention include preferred embodiments, such as AKRKSAA (SEQ ID NO:3), KKRASAA (SEQ ID NO:4), ARRASM (SEQ ID NO:5), AKRASM (SEQ ID NO:6), PKRASM (SEQ ID NO: 7), KKRKSAA (SEQ ID NO:8), KKRKSGL (SEQ ID NO:9), YNKRSTTI (SEQ ID NO:10), GVKRISGL (SEQ ID NO:11), APKRGSGK (SEQ

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