Title: Megakaryocytic protein tyrosine kinase I
Abstract: 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.
Patent Number: 6,908,984 Issued on 06/21/2005 to Ullrich, et al.
| Inventors:
|
Ullrich; Axel (Portola Valley, CA);
Gishizky; Mikhail (Palo Alto, CA);
Sures; Irmingard (Munich, DE)
|
| Assignee:
|
Sugen, Inc. (South San Francisco, CA);
Max-Planck-Gesellschaft zur Fonderung der Wissenschaften E.V. (Munich, DE)
|
| Appl. No.:
|
977261 |
| Filed:
|
October 16, 2001 |
| Current U.S. Class: |
530/350; 424/185.1; 424/192.1 |
| Intern'l Class: |
C07K 014/00; A61K039/00 |
| Field of Search: |
530/350
435/691,697,194
424/185.1,192.1
|
References Cited [Referenced By]
U.S. Patent Documents
| 5635177 | Jun., 1997 | Bennett.
| |
| 5834208 | Nov., 1998 | Sakano.
| |
| Foreign Patent Documents |
| WO 93/1520/1 | Aug., 1993 | WO.
| |
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|
Primary Examiner: Kunz; Gary
Assistant Examiner: Seharaseyon; Jegatheesan
Attorney, Agent or Firm: Burrous; Beth A., Foley & Lardner LLP
Parent Case Text
This application is a divisional application of U.S. application Ser. No. 08/426,509,
filed on Apr. 21, 1995, now U.S. Pat. No. 6,326,469, which is a continuation of
Ser. No. 08/232,545, filed on Apr. 22, 1994, now U.S. Pat. No. 6,506,578.
Claims
1. An isolated megakaryocyte protein tyrosine kinase 1, which has the amino acid
sequence depicted in SEQ ID NO. 2.
2. A fusion protein comprising megakaryocyte protein tyrosine kinase 1, which
has the amino acid sequence depicted in SEQ ID NO. 2 linked to a heterologous protein
or peptide sequence.
Description
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, non-receptor
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 or 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 of an immunodeficiency disease in humans,
(Arpala, E., et al.,
Cell 76:1-20, 1994). Gene knockout experiments in mice
suggest a role for src in the regulation of osteoclast function and bone remodeling
as these mice develop osteopetrosis. (Soriano et al.,
Cell 64:693-702, 1991
and Lowe et al., PNAS (in press)).
Megakaryocytes are large cells normally present in bone marrow and
spleen and are the progenitor cell for blood platelets. Megakaryocytes are associated
with such disease states as acute megakaryocytic leukemia (Lu et al.,
Cancer
Genet Cytogenet, 67(2):81-89 (1993) and Moody et al.,
Pediatr Radiol.
19(6-7):486-488 (1989)), a disease that is difficult to diagnose early and which
is characterized by aberrant proliferation of immature cells or "blasts"; myelofibrosis
(Smith et al.,
Crit Rev Oncol Hematol. 10(4):305-314 (1990) and Marino,
J. Am. Osteopath Assoc. 10:1323-1326 (1989)), an often fatal disease where
the malignant cell may be of megakaryocytic lineage and may be mediated by platelet
or megakaryocyte growth factors; acute megakaryocytic myelosis (Fohlmeister et
al.,
Haematologia 19(2):151-160 (1986)) a rapidly fatal disease characterized
by megakaryocytic proliferation and the appearance of immature megakaryocytes in
the circulation; and acute myelosclerosis (Butler et al.,
Cancer 49(12):2497-2499
(1982) and Bearman et al.,
Cancer 43(1):279-93 (1979)) a myeloproliferative
syndrome where the marrow is characterized by atypical megakaryocytes.
Platelets play a key role in the regulation of blood clotting and wound
healing, as well as being associated with such disease conditions as thrombocytopenia,
atherosclerosis, restenosis and leukemia. Several receptor tyrosine kinases have
been identified in human megakaryocytes including c-kit, blg and blk. (Hoffman,
H.,
Blood 74:1196-1212, 1989; Long, M. W.,
Stem Cells 11:33-40, 1993;
Zaebo, K. M., et al.,
Cell 63:213-224,1990). Cytoplasmic tyrosine kinases
of human megakaryocytic origin have also been reported. (Bennett et al.,
Journal
of Biological Chemistry 289(2):1068-1074, 1994; Lee et al.,
Gene 1-5,
1993; and Sakano et al.,
Oncogene 9:1155-1161 (1994)).
3. SUMMARY OF THE INVENTION
The present invention relates to novel, cytoplasmic tyrosine kinases isolated
from megakaryocytes (megakaryocyte kinases or MKKs) which are involved in cellular
signal transduction pathways. Particular MKKs described herein are referred to
as MKK1, MKK2, and MKK3. The complete nucleotide sequences encoding MKK1, MKK2,
and MKK3 are disclosed herein, and provide the basis for several aspects of the
invention hereinafter described.
The present invention is based, in part, upon the discovery that MKK1, MKK2,
and MKK3 have amino acid and structural homology, respectively, to the PTKs csk
(Brauninger et al.
Gene, 110:205-211 (1992) and Brauninger et al.,
Oncogene,
8:1365-1369 (1993)), atk/btk, tec and tsk (Vetrie et al.,
Nature 361:226-233
(1993); Mano et al.,
Oncogene 8:417-424 (1993) and Heyeck et al.,
PNAS
USA 90:669-673,1993, respectively) and fyn (Kawakami et al.
Mol. Cell. Bio.
6:4195-4201, 1986)).
The present invention also relates, in part, to nucleotide sequences and expression
vectors encoding MKKs. Also described herein are methods of treatment and diagnosis
of diseases resulting from abnormalities in signal transduction pathways in which
MKKs are involved.
The MKK sequences disclosed herein may be used to detect and quantify levels
of MKK mRNA in cells and furthermore for diagnostic purposes for detection of expression
of MKKs in cells. For example, an MKK sequence may be used in hybridization assays
of biopsied tissue to diagnose abnormalities in gene expression associated with
a transformed phenotype.
Also disclosed herein are methods of treatment of diseases or conditions associated
with abnormalities in signal transduction pathways in megakaryocytes. Such abnormalities
can result in, for example, under production of mature, differentiated cells, inappropriate
proliferation of immature cells or modulation of activity of other important cellular functions.
Anti-MKK antibodies may be used for diagnostic purposes for the detection
of MKKs in tissues and cells. Anti-MKK antibodies may also be used for therapeutic
purposes, for example, in neutralizing the activity of an MKK associated with a
signal transduction pathway.
Oligonucleotide sequences, including anti-sense RNA and DNA molecules
and ribozymes, designed to inhibit the translation of MKK mRNA, may be used therapeutically
in the treatment of disease states associated with aberrant expression of MKKs.
In a particular embodiment of the invention described by way of Example 9 herein,
an anti-MKK1 antisense molecule is used to inhibit MKK-1 protein synthesis resulting
in reduced megakaryocyte growth and differentiation.
Proteins, peptides and organic molecules capable of modulating activity
of MKKs may be used therapeutically in the treatment of disease states associated
with aberrant expression of MKKs. Alternatively, proteins, peptides and organic
molecules capable of modulating activity of MKKs may be used therapeutically to
enhance normal activity levels of MKKs. For example, small molecules found to stimulate
MKK1 activity in megakaryocytes may be used for ex vivo culturing of megakaryocytes
intended for autologous treatment of patients receiving chemotherapy or other therapies
which deplete megakaryoctyes or platelets, or in the treatment of thrombocytopenia.
4. BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1C. Human MKK1 nucliotide sequence (SEQ ID NO:1) and deduced amino
acid sequence (SEQ ID NO:2). Marked regions show the signal sequence, the SH2 and
SH3 domains, and the catalytic domain.
FIGS. 2A and 2B. Human MKK2 nucleotide sequence (SEQ ID NO:3) and deduced amino
acid sequence (SEQ ID NO:4). Marked regions show the signal sequence, the pleckstrin
homology domain (PH), the proline rich sequences following the PH domain, the SH2
and SH3 domains, and the catalytic domain.
FIG. 3. Human MKK3 nucleotide sequence (SEQ ID NO:5) and deduced amino
acid sequence (SEQ ID No:6). Marked regions show the signal sequence, the SH2 and
SH3 domains, and the catalytic domain.
FIG. 4. Expression of MKK1 and MKK2 in human and rodent cell lines.
FIG. 5. Immunoprecipitation (i.p.) of in vitro transcribed and translated
MKK1 and MKK2 proteins. Samples in lanes designated 1 through 9 are as follows:
1. MKK1 i.p. with anti-carboxy terminus MKK1 Ab, 2. and 3. MKK1 i.p. with anti-amino
terminus MKK1 Ab, 4. MKK1 i.p. with rabbit pre immune sera, 5. MKK2 i.p. with rabbit
pre immune sera, 6. and 7. MKK2 i.p. with anti-carboxy terminus MKK2 Ab, 8. MKK1
in vitro transcribed/translated protein without i.p., 9. MKK2 in vitro transcribed/translated
protein without i.p.
FIGS. 6A and 6B. FIGS. 6A-6B illustrate anti-sense MKK1 expression suppresses
AChE Production in primary murine bone marrow cultures. FIG. 6A illustrates AChE
production. FIG. 6B illustrates MKK1 protein expression.
FIG. 7. MKK2 and MKK3 autophosphorylate and transphosphorylate proteins
when expressed in bacteria. Lanes 2, 4, and 6 represent non-induced bacteria expressing
MKK1, MKK2, MKK3, respectively. Lanes 1, 3, and 5 represent induced bacteria expressing
MKK1, MKK2, MKK3, respectively.
FIG. 8. MKK expression constructs.
FIGS. 9A and 9B. Shared amino acid sequence homology of MKK1 SEQ ID NO: 2 and
csk SEQ ID NO: 7.
FIGS. 10A-10C SEQ ID NOS 4, 8-10 respectively, in order of appearance. Shared
amino acid sequence homology of MKK2 and atk/btk.
FIGS. 11A-11E SEQ ID NOS 6, 11-19, respectively, in order of appearance. Shared
amino acid sequence homology of MKK3 and src tyrosine kinase family members.
FIG. 12. FIG. 12 illustrates that the hyperexpression of MKK-1 in L-8057
cells grown in serum-free media inhibits cell growth of those cells as compared
to control L-8057 cells.
FIG. 13. FIG. 13 illustrates the stimulation of MKK-1 infected L-8057
cells and control L-8057 cells with rat stem cell factor and IL-3.
FIGS. 14A-14D. FIGS. 14A-14D illustrate the effect of tetradecanoyl phorbol
acetate ("TPA") on either control cells or cells that express MKK-1.
5. DETAILED DESCRIPTION
The present invention relates to novel, cytosolic megakaryocytic kinases referred
to herein as "MKKs", and in particular to megakaryocyte kinase 1 (MKK1), megakaryocyte
kinase 2 (MKK2), which are expressed in human megakaryocytic cell lines, and megakaryocyte
kinase 3 (MKK3).
As used herein, MKK is a term which refers to MKK1, MKK2 and MKK3 from any species,
including, bovine, ovine, porcine, equine, murine and preferably human, in naturally
occurring-sequence or in variant form, or from any source, whether natural, synthetic,
or recombinant. A preferred MKK variant is one having at least 80% amino acid homology,
a particularly preferred MKK variant is one having at least 90% sequence homology
and another particularly preferred MKK variant is one having at least 95% amino
acid homology to the naturally occurring MKK.
MKK1 is a cytosolic tyrosine kinase of molecular weight 58 kD, as determined
by SDS gel electrophoresis, having homology to the TK csk (Partanen, et al.,
Oncogene
6:2013-2018 (1991) and Nada et al.,
Nature 351:69-72 (1991)) in the
intervening sequences of its catalytic domain, the SH2 and SH3 domains, and other
non-catalytic regions and like csk, lacks regulatory phosphorylation sites corresponding
to c-src tyrosines 416 and 527. MKK1 also lacks an amino-terminal myristylation site.
Csk is a recently described novel cytoplasmic TK that seems to play a key role
in regulation of signal transduction in hematopoietic and neural development. For
example csk has been shown to negatively regulate members of the src family of
TKs, including c-src, lck, and fyn, through its ability to phosphorylate regulatory
tyrosines. (Bergman et al.,
The EMBO Journal 11(8)8:2919-2924 (1992) and
Sabe et al.,
Molecular and Cellular Biology 12(10):4706-4713 (1992)). Autero
et al., (
Molecular and Cellular Biology 14(2):1308-1321 (1994)) have reported
that csk positively regulates a phosphatase, CD45, that is key to T-cell activity.
Csk mediated phosphorylation of CD45 phosphotyrosine phosphatase (PTPase) caused
a several fold increase in its PTPase activity. Csk appears to play a role as a
regulator of the sequence of both phosphorylation and dephosphorylation events
culminating in cell activation and proliferation.
Defective expression of csk in mouse embryos results in defects in the
neural tube with subsequent death between day 9 and day 10 of gestation, with cells
derived from these embryos exhibiting an order of magnitude increase in activity
of src kinase (Nada et al.,
Cell 73:1125-1135 (1993)). Overexpression of
csk in transformed rat 3Y1 fibroblasts was shown to cause reversion to normal phenotypes
(Sabe et al.,
Molecular and Cellular Biology 12:4706-4713 (1992)).
MKK1 has 54% homology with csk at the amino acid level and structural similarity
to csk, i.e., the lack of regulatory phosphorylation sites and the lack of an amino-terminal
myristylation site. Experimental data, see Section 9, show that expression of human
anti-sense MKK1 sequences inhibits synthesis of murine MKK1, which inhibition is
associated with a reduction of proliferation of megakaryocytes in vitro. Based
upon the experimental data in Section 9 and amino acid and structural homology
with csk, MKK1 appears to play a regulatory role in the growth and differentiation
of megakaryocytes and perhaps neural tissues based on its expression in those tissues.
MKK2 is a novel cytosolic tyrosine kinase of molecular weight 78 kD, as determined
by SDS gel electrophoresis, having homology to the tec subfamily of TKs which also
incudes tsk and atk/btk. Like the tec subfamily, MKK2 lacks an amino-terminal site
for myristylation and has a putative pleckstrin homology binding domain located
5′ to the SH3 domain (Musacchio et al.,
TIBS 18:343-348 (1993)).
The pleckstrin homology (PH) domain has been found in a number of proteins with
diverse cellular functions and is abundant in proteins involved in signal transduction
pathways. Musacchio et al., supra suggest that the PH domain may be involved in
molecular recognition similarly to SH2 and SH3 domains.
The tec family of tyrosine kinases appear to play roles in cellular differentiation
and include family members tec, a kinase which may be specifically involved in
the cell growth of hepatocytes or hepatocarcinogenesis (Mano et al., supra); tsk,
which may play a role in early T-lymphocyte differentiation (Heyek et al.,
PNAS
USA 90:669-673 (1993)) and atk/btk. Aberrant expression of atk/btk has been
shown to be responsible for X-linked agammaglobulinemia (XLA), a human disease
resulting from a developmental block in the transition from pre-B cells to mature
B cells (Ventrie, D. et al., supra).
MKK2 has 50% homology to atk/btk at the amino acid level and structural similarity
to tec family members, i.e., the presence of the SH2, SH3 and PH domains and the
lack of an amino-terminal site for myristylation and the carboxyl site of tyrosine
phosphorylation found in family members. Based upon the amino acid homology and
structural similarity to tec family members which play roles in cellular differentiation,
MKK2 may play a role in the differentiation of megakaryoctyes.
MKK3 is a novel cytosolic tyrosine kinase of molecular weight 58kD, as determined
by SDS gel electrophoresis, having homology to the TK fyn. MKK3 does not have a
myristylation sites. MKK3 does have a putative regulatory cite at tyr 387 but the
surrounding 12 amino acids are not identical with other members of the src subfamily
that share highly conserved sequences in this region. MKK3 has 47% homology with
fyn at the amino acid level.
The fyn gene was originally characterized in normal human fibroblast and endothelial
cells, but it is also expressed in a variety of other cell types. Alternative splicing
of fyn has been shown to yield two distinct transcripts, both coding for enzymatically
active forms of the kinases.
MKK sequences could be used diagnostically to measure expression of MKKs in disease
states, such as for example leukemia, where abnormal proliferation of immature
myeloid cells occurs, or where abnormal differentiation of megakaryocytes occurs.
MKKs could also be used therapeutically in the treatment of disease states involving
abnormal proliferation or differentiation through interruption of signal transduction
by modulation of protein tyrosine kinases.
The nucleotide and deduced amino acid sequence of human MKK1, MKK2, and MKK3
are shown in FIGS. 1A-1C (SEQ ID NOS 1-2),
2A-
2B (SEQ ID NOS 3-4)
and
3 (SEQ ID NOS 5-6), respectively. FIGS. 9A-9B (SEQ ID NOS 2 and 7, respectively,
in order of appearance),
10A-
10C (SEQ ID NOS 4, 8-10, respectively,
in order of appearance) and
11A-
11E (SEQ ID NOS 6, 11-19, respectively,
in order of appearance) show the shared sequence homology between MKKs and related
tyrosine kinases.
5.1 The MKK Coding Sequences
The nucleotide coding sequence and deduced amino acid sequence of the human MKK1,
MKK2, and MKK3 genes are depicted in FIGS. 1A-11C (SEQ ID NOS 1-2),
2A-
2B
(SEQ ID NOS 3-4) and
3 (SEQ ID NOS 5-6), respectively. In accordance with
the invention, any nucleotide sequence which encodes the amino acid sequence of
an MKK gene product can be used to generate recombinant molecules which direct
the expression of an MKK.
In a specific embodiment described herein, the human MKK1, MKK2, and MKK3 genes
were isolated by performing polymerase chain reactions (PCR) in combination with
two degenerate oligonucleotide primer pools that were designed on the basis of
highly conserved sequences within the kinase domain of receptor tyrosine kinases
corresponding to the amino acid sequence HRDLAA (residues 350-355 of SEQ ID NO:
2) (sense primer) and SDVWSF/Y (SEQ ID NO:24) (antisense primer) (Hanks et al.,
1988). The MKK cDNAs were synthesized by reverse transcription of poly-A RNA from
the human K-562 cell line, ATCC accession number CCL 243, or from the Meg 01 cell
line, (Ogura et al.,
Blood 66: 1384 (1985)).
The PCR fragments were used to screen a lambda gt11 library of human fetal brain.
For each individual MKK, several overlapping clones were identified. The composite
of the cDNA clones for MKK1, MKK2, and MKK3 are depicted in FIGS. 1A-1C (SEQ ID
NOS 1-2),
2A-
2B (SEQ ID NOS 3-4), and
3 (SEQ ID NOS 5-6), respectively.
Further characterization of the individual MKKs is found infra.
5.2. Expression of MKK
In accordance with the invention, MKK polynucleotide sequences which encode MKKs,
peptide fragments of MKKs, MKK fusion proteins or functional equivalents thereof,
may be used to generate recombinant DNA molecules that direct the expression of
MKK protein, MKK peptide fragment, fusion proteins or a functional equivalent thereof,
in appropriate host cells. Such MKK polynucleotide sequences, as well as other
polynucleotides which selectively hybridize to at least a part of such MKK polynucleotides
or their complements, may also be used in nucleic acid hybridization assays, Southern
and Northern blot analyses, etc.
Due to the inherent degeneracy of the genetic code, other DNA sequences which
encode substantially the same or a functionally equivalent amino acid sequence,
may be used in the practice of the invention for the cloning and expression of
the MKK protein. Such DNA sequences include those which are capable of hybridizing
to the human MKK sequence under stringent conditions. The phrase "stringent conditions"
as used herein refers to those hybridizing conditions that (1) employ low ionic
strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium
citrate/0.1% SDS at 50° C.; (2) employ during hybridization a denaturing agent
such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at
pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50%
formamide, 5×SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5×Denhardt's
solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate
at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.
Altered DNA sequences which may be used in accordance with the invention
include deletions, additions or substitutions of different nucleotide residues
resulting in a sequence that encodes the same or a functionally equivalent gene
product. The gene product itself may contain deletions, additions or substitutions
of amino acid residues within an MKK sequence, which result in a silent change
thus producing a functionally equivalent MKK. Such amino acid substitutions may
be made on the basis of similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipatic nature of the residues involved. For example,
negatively charged amino acids include aspartic acid and glutamic acid; positively
charged amino acids include lysine and arginine; amino acids with uncharged polar
head groups having similar hydrophilicity values include the following: leucine,
isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine;
phenylalanine, tyrosine.
The DNA sequences of the invention may be engineered in order to alter an MKK
coding sequence for a variety of ends including but not limited to alterations
which modify processing and expression of the gene product. For example, mutations
may be introduced using techniques which are well known in the art, e.g., site-directed
mutagenesis, to insert new restriction sites, to alter glycosylation patterns,
phosphorylation, etc.
In another embodiment of the invention, an MKK or a modified MKK sequence may
be ligated to a heterologous sequence to encode a fusion protein. For example,
for screening of peptide libraries for inhibitors of MKK activity, it may be useful
to encode a chimeric MKK protein expressing a heterologous epitope that is recognized
by a commercially available antibody. A fusion protein may also be engineered to
contain a cleavage site located between an MKK sequence and the heterologous protein
sequence, so that the MKK may be cleaved away from the heterologous moiety.
In an alternate embodiment of the invention, the coding sequence of an MKK could
be synthesized in whole or in part, using chemical methods well known in the art.
See, for example, Caruthers et al., 1980
, Nuc. Acids Res. Symp. Ser. 7:215-233;
Crea and Horn, 180
, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers,
1980
, Tetrahedron Letters 21:719; and Chow and Kempe, 1981
, Nuc. Acids
Res. 9(12):2807-2817. Alternatively, the protein itself could be produced using
chemical methods to synthesize an MKK amino acid sequence in whole or in part.
For example, peptides can be synthesized by solid phase techniques, cleaved from
the resin, and purified by preparative high performance liquid chromatography.
(e.g., see Creighton, 1983
, Proteins Structures And Molecular Principles,
W. H. Freeman and Co., N.Y. pp. 50-60). The composition of the synthetic peptides
may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation
procedure; see Creighton, 1983
, Proteins, Structures and Molecular Principles,
W. H. Freeman and Co., N.Y., pp. 34-49.
In order to express a biologically active MKK, the nucleotide sequence coding
for MKK, or a functional equivalent, is inserted into an appropriate expression
vector, i.e., a vector which contains the necessary elements for the transcription
and translation of the inserted coding sequence. The MKK gene products as well
as host cells or cell lines transfected or transformed with recombinant MKK expression
vectors can be used for a variety of purposes. These include but are not limited
to generating antibodies (i.e., monoclonal or polyclonal) that competitively inhibit
activity of an MKK and neutralize its activity. Anti-MKK antibodies may be used
in detecting and quantifying expression of an MKK in cells and tissues.
5.3. Expression Systems
Methods which are well known to those skilled in the art can be used to construct
expression vectors containing an MKK coding sequence and appropriate transcriptional/translational
control signals. These methods include in vitro recombinant DNA techniques, synthetic
techniques and in vivo recombination/genetic recombination. See, for example, the
techniques described in Maniatis et al., 1989
, Molecular Cloning A Laboratory
Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989
, Current
Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.
A variety of host-expression vector systems may be utilized to express an MKK
coding
sequence. These include but are not limited to microorganisms such as bacteria
transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing an MKK coding sequence; yeast transformed with recombinant yeast
expression vectors containing an MKK coding sequence; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus) containing an MKK
coding sequence; plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed
with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an MKK
coding sequence; or animal cell systems. The expression elements of these systems
vary in their strength and specificities. Depending on the host/vector system utilized,
any of a number of suitable transcription and translation elements, including constitutive
and inducible promoters, may be used in the expression vector. For example, when
cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ,
plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning
in insect cell systems, promoters such as the baculovirus polyhedrin promoter may
be used; when cloning in plant cell systems, promoters derived from the genome
of plant cells (e.g., heat shock promoters; the promoter for the small subunit
of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant
viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV)
may be used; when cloning in mammalian cell systems, promoters derived from the
genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be
used; when generating cell lines that contain multiple copies of an MKK DNA, SV40-,
BPV- and EBV-based vectors may be used with an appropriate selectable marker.
In bacterial systems a number of expression vectors may be advantageously selected
depending upon the use intended for the MKK expressed. For example, when large
quantities of MKK1 are to be produced for the generation of antibodies, vectors
which direct the expression of high levels of fusion protein products that are
readily purified may be desirable. Such vectors include but are not limited to
the
E. coli expression vector pUR278 (Ruther et al., 1983
, EMBO J.
2:1791), in which the MKK1 coding sequence may be ligated into the vector in frame
with the lac Z coding region so that a hybrid AS-lac Z protein is produced; pIN
vectors (Inouye & Inouye, 1985
, Nucleic acids Res. 13:3101-3109; Van Heeke
& Schuster, 1989
, J. Biol. Chem. 264:5503-5509); and the like. PGEX vectors
may also be used to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose beads followed
by elution in the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide
of interest can be released from the GST moiety.
In yeast, a number of vectors containing constitutive or inducible promoters
may
be used. For a review see,
Current Protocols in Molecular Biology, Vol.
2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13;
Grant et al., 1987
, Expression and Secretion Vectors for Yeast, in Methods
in Enzymology, Ed. Wu & Grossman, 1987, Acad. Press, N.Y. 153:516-544; Glover,
1986
, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987,
Heterologous Gene Expression in Yeast,
Methods in Enzymology, Eds. Berger
& Kimmel, Acad. Press, N.Y. 152:673-684; and
The Molecular Biology of the Yeast
Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols.
I and II.
In cases where plant expression vectors are used, the expression of an MKK-coding
sequence may be driven by any of a number of promoters. For example, viral promoters
such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984
, Nature
310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987
,
EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the
small subunit of RUBISCO (Coruzzi et al., 1984
, EMBO J. 3:1671-1680; Broglie
et al., 1984
, Science 224:838-843); or heat shock promoters, e.g., soybean
hsp17.5-E or hsp17.3-B (Gurley et al., 1986
, Mol. Cell. Biol. 6:559-565)
may be used. These constructs can be introduced into plant cells using Ti plasmids,
Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation,
etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988
,
Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp.
421-463; and Grierson & Corey, 1988
, Plant Molecular Biology, 2d Ed., Blackie,
London, Ch. 7-9.
An alternative expression system which could be used to express an MKK is an
insect
system. In one such system,
Autographa californica nuclear polyhidrosis
virus (AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. An MKK coding sequence may be cloned into non-essential
regions (for example the polyhedrin gene) of the virus and placed under control
of an AcNPV promoter (for example, the polyhedrin promoter). Successful insertion
of an MKK coding sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous
coat coded for by the polyhedrin gene). These recombinant viruses are then used
to infect
Spodoptera frugiperda cells in which the inserted gene is expressed.
(e.g., see Smith et al., 1983
, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051).
In mammalian host cells, a number of viral based expression-systems may be utilized.
In cases where an adenovirus is used as an expression vector, an MKK coding sequence
may be ligated to an adenovirus transcription/translation control complex, e.g.,
the late promoter and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion
in a non-essential region of the viral genome (e.g., region E1 or E3) will result
in a recombinant virus that is viable and capable of expressing an MKK in infected
hosts. (e.g., See Logan & Shenk, 1984
, Proc. Natl. Acad. Sci. (
USA)
81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used. (See, e.g.,
Mackett et al., 1982
, Proc. Natl. Acad. Sci. (
USA) 79:7415-7419;
Mackett et al., 1984
, J. Virol. 49:857-864; Panicali et al., 1982
, Proc.
Natl. Acad. Sci. 79:4927-4931).
Specific initiation signals may also be required for efficient translation
of an inserted MKK coding sequences. These signals include the ATG initiation codon
and adjacent sequences. In cases where an entire MKK gene, including its own initiation
codon and adjacent sequences, is inserted into the appropriate expression vector,
no additional translational control signals may be needed. However, in cases where
only a portion of an MKK coding sequence is inserted, exogenous translational control
signals, including the ATG initiation codon, must be provided. Furthermore, the
initiation codon must be in phase with the reading frame of an MKK coding sequence
to ensure translation of the entire insert. These exogenous translational control
signals and initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate
transcription enhancer elements, transcription terminators, etc. (see Bittner et
al., 1987
, Methods in Enzymol. 153:516-544).
In addition, a host cell strain may be chosen which modulates the expression
of
the inserted sequences, or modifies and processes the gene product in the specific
fashion desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein products may be important for the function of the protein.
Different host cells have characteristic and specific mechanisms for the post-translational
processing and modification of proteins. Appropriate cells lines or host systems
can be chosen to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells which possess the cellular
machinery for proper processing of the primary transcript, glycosylation, and phosphorylation
of the gene product may be used. Such mammalian host cells include but are not
limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.
For long-term, high-yield production of recombinant proteins, stable expression
is preferred. For example, cell lines which stably express an MKK may be engineered.
Rather than using expression vectors which contain viral origins of replication,
host cells can be transformed with MKK DNA controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following the introduction
of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci which in turn
can be cloned and expanded into cell lines. This method may advantageously be used
to engineer cell lines which express an MKK.
A number of selection systems may be used, including but not limited to the herpes
simplex virus thymidine kinase (Wigler et al., 1977
, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962
, Proc. Natl. Acad. Sci.
USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980
,
Cell 22:817) genes can be employed in tk
-;, hgprt
-; or
aprt
-; cells, respectively. Also, antimetabolite resistance can be used
as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler
et al., 1980
, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981
, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic
acid (Mulligan & Berg, 1981),
Proc. Natl. Acad. Sci. USA 78:2072); neo,
which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,
1981
, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin
(Santerre et al., 1984
, Gene 30:147). Recently, additional selectable genes
have been described, namely trpB, which allows cells to utilize indole in place
of tryptophan; hisD, which allows cells to utilize histinol in place of histidine
(Hartman & Mulligan, 1988
, Proc. Natl. Acad. Sci. USA 85:8047); and ODC
(ornithine decarboxylase) which confers resistance to the ornithine decarboxylase
inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987
, In: Current
Communications in Molecular Biology, Cold Spring Harbor Laboratory, Ed.).
5.4. Identification of Transfectants or Transformants that Express the MKK
The host cells which contain the coding sequence and which express the biologically
active gene product may be identified by at least four general approaches; (a)
DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene
functions; (c) assessing the level of transcription as measured by the expression
of MKK mRNA transcripts in the host cell; and (d) detection of the gene product
as measured by immunoassay or by its biological activity.
In the first approach, the presence of the MKK coding sequence inserted in the
expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes
comprising nucleotide sequences that are homologous to the MKK coding sequence,
respectively, or portions or derivatives thereof.
In the second approach, the recombinant expression vector/host system can be
identified
and selected based upon the presence or absence of certain "marker" gene functions
(e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate,
transformation phenotype, occlusion body formation in baculovirus, etc.). For example,
if the MKK1 coding sequence is inserted within a marker gene sequence of the vector,
recombinant cells containing the MKK1 coding sequence can be identified by the
absence of the marker gene function. Alternatively, a marker gene can be placed
in tandem with an MKK sequence under the control of the same or different promoter
used to control the expression of the MKK coding sequence. Expression of the marker
in response to induction or selection indicates expression of the MKK coding sequence.
In the third approach, transcriptional activity for an MKK coding region can
be
assessed by hybridization assays. For example, RNA can be isolated and analyzed
by Northern blot using a probe homologous to an MKK coding sequence or particular
portions thereof. Alternatively, total nucleic acids of the host cell may be extracted
and assayed for hybridization to such probes.
In the fourth approach, the expression of an MKK protein product can be assessed
immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation,
enzyme-linked immunoassays and the like.
5.5. Uses of MKK and Engineered Cell Lines
Megakaryocytes, the progenitor cell for blood platelets, and platelets
are associated with disease states involving aberrant proliferation or differentiation
of such cells, such as acute megakaryocytic leukemia, acute megakaryocytic myelosis
and thrombocytopenia. MKKs appear to play a role in the growth and differentiation
of megkaryocytes, therefore inhibitors of MKKs may be used therapeutically for
the treatment of diseases states resulting from aberrant growth of megakaryocytes
or platelets. Alternatively, enhancers of MKKs may be used therapeutically to stimulate
the proliferation of megakaryocytes in such applications as, for example, ex vivo
culturing of megakaryocytes intended for autologous cell therapy in individuals
receiving chemotherapy or other therapies which deplete megakaryocytes or platelets
or in treating thrombocytopenia caused by other conditions.
In an embodiment of the invention, an MKK and/or cell line that expresses an
MKK
may be used to screen for antibodies, peptides, or other molecules that act as
agonists or antagonists of MKK through modulation of signal transduction pathways.
For example, anti-MKK antibodies capable of neutralizing the activity of MKK may
be used to inhibit an MKK associated signal transduction pathway. Such antibodies
can act intracellularly utilizing the techniques described in Marasco et al.(
PNAS
90:7889-7893 (1993) for example or through delivery by liposomes. Alternatively,
screening of organic or peptide libraries with recombinantly expressed MKK protein
or cell lines expressing MKK protein may be useful for identification of therapeutic
mol
