Megakaryocytic protein tyrosine kinases

Details Megakaryocytic protein tyrosine kinases Megakaryocytic protein tyrosine kinases

1995-04-21

2001-12-04

Stucker, Jeffrey (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

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

C530S350000, C435S069100, C435S069700, C435S194000

Reexamination Certificate

active

06326469

ABSTRACT:

TABLE OF CONTENTS
1. Introduction
2. Background
3. Summary of the Invention
4
. Brief Description of the Figures
5
. Detailed Description
5.1. The MKK Coding Sequences
5.2. Expression of MKK
5.3. Expression Systems
5.4. Identification of Transfectants or Transformants that Express the MKK
5.5. Uses of MKK and Engineered Cell Lines
5.5.1. Antibody Production and Screening
5.5.2. Screening of Peptide Library with MKK or MKK Engineered Cell Lines
5.5.3. Screening of Organic Compounds with MKK Protein or Engineered Cell Lines
5.6. Uses of MKK Polynucleotide
5.6.1. Diagnostic Uses of an MKK Polynucleotide
5.6.2. Therapeutic Uses of an MKK Polynucleotide
6. Examples: Cloning and Characterization of MKK1
6.1. cDNA Cloning, MKK Expression and MKK Characterization
6.1.1. Full-length cDNA Cloning
6.1.2. MKK Expression
6
.
1
.
3
. RNA Blot Analysis of MKKs
7. Example: Autophosphorylation of MKK2 and MKK3
8. Example: Production of Anti-MKK Antibodies and Immunoprecipitation of MKK
9. Example: Expression of MKK1 Anti-sense Sequences
10. Example: MKKR Protein Tyrosine Kinase Activity
10.1. Materials and Methods .
10
.
2
. Results
11. Example: Biological Activity of MKK1
11.1. Materials and Methods
11.1.1. Retroviral Infection of L-8057 cells
11.1.2. Cell Growth Measurement of MKK1 Expressing L-8057 Cells
11.1.3. Growth Factor Response of MKK1 Expressing L-8057 Cells
11.2. Results
11.2.1. Cell Growth
11.2.2. Cytokine Stimulation
11.2.3. Cell Differentiation
WHAT IS CLAIMED
ABSTRACT
1. Introduction
The present invention relates to novel cytoplasmic tyrosine kinases isolated from megakaryocytes (megakaryocyte kinases or MKKs) which are involved in cellular signal transduction pathways and to the use of these novel proteins in the diagnosis and treatment of disease.
The present invention further relates to specific megakaryocyte kinases, designated MKK1, MKK2 and MKK3, and their use as diagnostic and therapeutic agents.
2. Background
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. These processes include, but are not limited to, cell proliferation, differentiation and survival. Many tyrosine kinases are expressed in postmitotic, fully differentiated cells, particularly in the case of hematopoietic cells, and it seems likely that these proteins are involved in specialized cellular functions that are specific for the cell types in which they are expressed. (Eiseman, E. and J. B. Bolen,
Cancer Cells
2(10):303-310, 1990). A central feature of signal transduction is the reversible phosphorylation of certain proteins. (for reviews, see Posada, J. and Cooper, J. A., 1992
, Mol. Biol. Cell
3:583-392; Hardie, D. G., 1990
, Symp. Soc. Exp. Biol
. 44:241-255). The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs), which function to phosphorylate proteins, and tyrosine phosphatases (TPs), which function to dephosphorylate proteins. Normal cellular function requires a delicate balance between the activities of these two types of enzyme.
Phosphorylation of cell surface tyrosine kinases, stimulates a physical association of the activated receptor with intracellular target molecules. Some of the target molecules are in turn phosphorylated. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal transducer proteins.
The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, S. A.,
Science
254:1146-1153, 1991; Schlessinger, J.
Trends Biochem. Sci
. 13:443-447, 1988; and Ullrich, A., and Schlessinger,
J. Cell
61:203-212, 1990.
Receptor tyrosine kinases are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. The intracellular, cytoplasmic, nonreceptor protein tyrosine kinases may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane domain. Bolen (
Oncogene
, vol. 8, pgs. 2025-2031 (1993)) reports that 24 individual protein tyrosine kinases comprising eight different families of non-receptor protein tyrosine kinases have been identified: Abl/Arg; Jak1/Jak2/Tyk2; Fak; Fes/Fps; Syk/Zap; Tsk/Tec/Atk; Csk; and the Src group, which includes the family members Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. All of the non-receptor protein tyrosine kinases are thought to be involved in signaling pathways that modulate growth and differentiation. Bolen, supra, suggests that half of the nonreceptor protein tyrosine kinases have demonstrated oncogenic potential and half appear to be primarily related to suppressing the activity of Src-related protein kinases and could be classified as anti-oncogenes.
While distinct in their overall molecular structure, each member of a given morphotypic family of cytoplasmic protein tyrosine kinases shares sequence homology in certain non-catalytic domains in addition to sharing sequence homology in the catalytic kinase domain. Examples of defined non-catalytic domains include the SH2 (SRC homology domain 2; Sadowski, I et al.,
Mol. Cell. Biol
. 6:4396-4408; Kock, C. A. et al., 1991
, Science
252:668-674) domains, SH3 domains (Mayer, B. J. et al., 1988
, Nature
332:269-272) and PH domains (Musacchio et al.,
TIBS
18:343-348 (1993). These non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson, T. and Gish, G., 1992
, Cell
71:359-362).
While the metabolic roles of cytoplasmic protein tyrosine kinases are less well understood than that of the receptor-type protein tyrosine kinases, significant progress has been made in elucidating some of the processes in which this class of molecules is involved. For example, members of the src family, lck and fyn, have been shown to interact with CD4/CD8 and the T cell receptor complex, and are thus implicated in T cell activation, (Veillette, A. Davidson, D., 1992
, TIG
8:61-66). Some cytoplasmic protein tyrosine kinases have been linked to certain phases of the cell cycle (Morgan, D. O. et al., 1989
, Cell
57:775-786; Kipreos, E. T. et al., 1990
, Science
248:217-220; Weaver et al., 1991
, Mol. Cell. Biol
. 11:4415-4422), and cytoplasmic protein tyrosine kinases have been implicated in neuronal and hematopoietic development (Maness, P., 1992
, Dev. Neurosci
14:257-270 and Rawlings et al.,
Science
261:358-361 (1993)). Deregulation of kinase activity through mutation or overexpression is a well-established mechanism underlying cell transformation (Hunter et al., 1985, supra; Ullrich et al., supra).
A variety of cytoplasmic tyrosine kinases are expressed in, and may have important functions in, hematopoietic cells including src, lyn, fyn, blk, lck, csk and hck. (Eisenian, E. and J. B. Bolen,
Cancer Cells
2(10):303-310, 1990). T-cell activation, for example, is associated with activation of lck. The signaling activity of lyn may be stimulated by binding of allergens to IgE on the surface of basophils. (Eisenian, supra).
Abnormalities in tyrosine kinase regulated signal transduction pathways can result in a number of disease states. For example, mutations in the cytoplasmic tyrosine kinase atk (also called btk) are responsible for the x-linked agammaglobulinemia, (Ventrie, D., et al.,
Nature
361:226-23, 1993). This defect appears to prevent the normal differentiation of pre-B cells to mature circulating B cells and results in a complete lack of serum immunoglobulins of all isotypes. The cytoplasmic tyrosine kinase Zap-70 has been suggested as indispensable for the development of CD8 single-positive T cells as well as for signal transduction and function of single-positive CD4 T cells, and lack of this protein leads to an immunodeficiency disease in humans, (Arpala, E.,

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