Control of TGFbeta signaling by km23 superfamily members

A method of determining the presence or absence of a lesion in a patient specimen which is characterized by a specific alteration in sequence of a km23 nucleic acid or polypeptide form, the method comprising:

• (a) providing a km23 genomic nucleic acid form from a patient specimen;
• (b) providing a first pair of PCR primers which span exon 3 of said km23 genomic nucleic acid form;
• and
• (c) performing a first round of nested PCR amplification of a region of said km23 genomic nucleic acid form comprising exon 3 using said first pair of primers under suitable conditions to obtain a first PCR product;
• (d) providing a second pair of PCR primers capable of binding within said first PCR product;
• (e) performing a second round of nested PCR amplification using said first PCR product as the reaction template under suitable conditions to obtain a second PCR product;
• and
• (f) sequencing and aligning said second PCR product with a wild-type km23 nucleic acid form to determine a specific alteration in sequence.

A method of determining the presence or absence of a lesion in a patient specimen which is characterized by a specific alteration in sequence of a km23 nucleic acid or polypeptide form, the method comprising:

• (a) providing a km23 genomic nucleic acid form from a patient specimen;
• (b) providing a first pair of PCR primers which span exon 2 of said km23 genomic nucleic acid form;
• and
• (c) performing a first round of nested PCR amplification of a region of said km23 genomic nucleic acid form comprising exon 2 using said first pair of primers under suitable conditions to obtain a first PCR product;
• (d) providing a second pair of PCR primers capable of binding within said first PCR product;
• (e) performing a second round of nested PCR amplification using said first PCR product as the reaction template under suitable conditions to obtain a second PCR product;
• and
• (f) sequencing and aligning said second PCR product with a wild-type km23 nucleic acid form to determine a specific alteration in sequence.

A method of determining the presence or absence of a lesion in a patient specimen characterized by an specific alteration in sequence of a km23 nucleic acid or polypeptide form, the method comprising:

• (a) providing a km23 genomic nucleic acid form from a patient specimen;
• (b) providing a first pair of PCR primers which span exon 4 of said km23 genomic nucleic acid form;
• and
• (c) performing a first round of nested PCR amplification of a region of said km23 genomic nucleic acid form comprising exon 4 using said first pair of primers under suitable conditions to obtain a first PCR product;
• (d) providing a second pair of PCR primers capable of binding within said first PCR product;
• (e) performing a second round of nested PCR amplification using said first PCR product as the reaction template under suitable conditions to obtain a second PCR product;
• and
• (f) sequencing and aligning said second PCR product with a wild-type km23 nucleic acid form to determine a specific alteration in sequence.

A method for screening for the presence or absence of a lesion in a patient specimen which is characterized by a specific alteration in a km23 nucleic acid or polypeptide form, the method comprising:

• (i) providing a km23 nucleic acid form from a patient specimen;
• and
• (ii) detecting the presence of a specific alteration in a km23 nucleic acid form relative to the wild-type km23 nucleic acid form, which alteration results in one of the group consisting of:
• (a) a mutant km23 polypeptide having a Gly at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (b) a mutant km23 polypeptide having a Val at the amino acid corresponding to amino acid number 89 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (c) a mutant km23 polypeptide having a Ile at the amino acid corresponding to amino acid number 46 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (d) a mutant km23 polypeptide having a Ser at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (e) a mutant km23 polypeptide having an Ala at the amino acid corresponding to amino acid number 4 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (f) a mutant km23 polypeptide having an Ala, Asp, or Glu at the amino acid corresponding to amino acid number 32 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (g) a mutant km23 polypeptide having a Glu or Asp at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (h) a mutant km23 polypeptide having a Ala, Glu, or Asp at the amino acid corresponding to amino acid number 73 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (i) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 7 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (j) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 33 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (k) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (l) a mutant km23 polypeptide having an Ala or Glu at the amino acid corresponding to amino acid number 13 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (m) a km23 polypeptide comprising SEQ ID NO:
• 1 or SEQ ID NO:
• 2, except that the polypeptide lacks amino acids corresponding to amino acids 27-82 in SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (n) a km23 polypeptide comprising the amino acid sequence corresponding to SEQ ID NO:
• 6.

A method of screening for a therapeutic agent that inhibits or stimulates the interaction of a km23 polypeptide form with a specific binding protein, the method comprising:

• (a) providing drug-inducible km23 expressing cells, wherein said expressed km23 is a wild-type form or an altered form thereof;
• (b) treating the cells of (a) with an effective amount of a therapeutic candidate so as to alter the growth inhibitory effect of km23;
• and
• (c) measuring the effect of the candidate on the cells with a cell growth assay.

A method of screening for a therapeutic agent that modulates the interaction of a km23 polypeptide form with the dynein intermediate chain, the method comprising:

• (a) providing a fusion protein comprising a dynein intermediate chain polypeptide labeled with a first fluorophore;
• (b) providing a fusion protein comprising a km23 polypeptide form labeled with a second fluorophore;
• (c) incubating components (a) and (b) in the presence and absence of an effective amount of a therapeutic candidate under suitable conditions to allow protein interaction between components (a) and (b);
• and
• (d) detecting the effect of said candidate on said interaction by comparing the fluorescence spectra for each of said fluorescently labeled polypeptides in the presence and absence of said candidate.

An isolated or recombinant km23 polypeptide comprising SEQ ID NO:

• 1 or SEQ ID NO:
• 2, except that the polypeptide lacks amino acids corresponding to amino acids 27-82 in SEQ ID NO:
• 1 or SEQ ID NO:
• 2.

A mutant km23 polypeptide selected from the group consisting of:

• (a) a mutant km23 polypeptide having a Gly at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (b) a mutant km23 polypeptide having a Val at the amino acid corresponding to amino acid number 89 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (c) a mutant km23 polypeptide having a Ile at the amino acid corresponding to amino acid number 46 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (d) a mutant km23 polypeptide having a Ser at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (e) a mutant km23 polypeptide having an Ala at the amino acid corresponding to amino acid number 4 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2.

A mutant km23 polypeptide selected from the group consisting of:

• (a) a mutant km23 polypeptide having an Ala, Asp, or Glu at the amino acid corresponding to amino acid number 32 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (b) a mutant km23 polypeptide having a Glu or Asp at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (c) a mutant km23 polypeptide having a Ala, Glu, or Asp at the amino acid corresponding to amino acid number 73 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (d) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 7 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (e) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 33 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (f) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (g) a mutant km23 polypeptide having an Ala or Glu at the amino acid corresponding to amino acid number 13 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2.

A cloned mutant km23 nucleic acid form, wherein said mutant is characterized by a mutation of at least one nucleotide base relative to a wild-type km23 nucleic acid form which mutation results in a mutant km23 polypeptide selected from the group consisting of:

• (a) a mutant km23 polypeptide having a Gly at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (b) a mutant km23 polypeptide having a Val at the amino acid corresponding to amino acid number 89 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (c) a mutant km23 polypeptide having a Ile at the amino acid corresponding to amino acid number 46 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (d) a mutant km23 polypeptide having a Ser at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (e) a mutant km23 polypeptide having an Ala at the amino acid corresponding to amino acid 4 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2

A cloned mutant km23 nucleic acid form wherein said mutant is characterized by mutation of at least one nucleotide base relative to a wild-type km23 nucleic acid form which mutation results in a mutant km23 polypeptide selected from the group consisting of:

• (a) a mutant km23 polypeptide having an Ala, Asp, or Glu at the amino acid corresponding to amino acid number 32 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (b) a mutant km23 polypeptide having a Glu or Asp at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (c) a mutant km23 polypeptide having a Ala, Glu, or Asp at the amino acid corresponding to amino acid number 73 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (d) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 7 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (e) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 33 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (f) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (g) a mutant km23 polypeptide having an Ala or Glu at the amino acid corresponding to amino acid number 13 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2.

A cloned km23 nucleic acid form comprising a nucleotide sequence encoding a km23 polypeptide comprising SEQ ID NO:

• 1 or SEQ ID NO:
• 2, except that the polypeptide lacks amino acids corresponding to amino acids 27-82 in SEQ ID NO:
• 1 or SEQ ID NO:
• 2.

An expression vector capable of expressing in a host cell a km23 polypeptide or mutant km23 polypeptide selected from the group consisting of:

• (a) a mutant km23 polypeptide having a Gly at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (b) a mutant km23 polypeptide having a Val at the amino acid corresponding to amino acid number 89 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (c) a mutant km23 polypeptide having a Ile at the amino acid corresponding to amino acid number 46 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (d) a mutant km23 polypeptide having a Ser at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (e) a mutant km23 polypeptide having an Ala at the amino acid corresponding to amino acid number 4 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2.
• (f) a mutant km23 polypeptide having an Ala, Asp, or Glu at the amino acid corresponding to amino acid number 32 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (g) a mutant km23 polypeptide having a Glu or Asp at the amino acid corresponding to amino acid number 55 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (h) a mutant km23 polypeptide having a Ala, Glu, or Asp at the amino acid corresponding to amino acid number 73 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (i) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 7 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 33 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• (k) a mutant km23 polypeptide having a Ala, Asp, or Glu at the amino acid corresponding to amino acid number 56 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• (l) a mutant km23 polypeptide having an Ala or Glu at the amino acid corresponding to amino acid number 13 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2.
• (m) an isolated or recombinant km23 polypeptide comprising SEQ ID NO:
• 1 or SEQ ID NO:
• 2, except that the polypeptide lacks amino acids corresponding to amino acids 27-82 in SEQ ID NO:
• 1 or SEQ ID NO:
• 2.
• (n) an isolated or recombinant km23 polypeptide comprising the amino acid sequence corresponding to SEQ ID NO:
• 6.

A kit for diagnostic or prognostic screening of a km23 nucleic acid sample, wherein the kit comprises:

• (a) a first pair of PCR primers comprising SEQ ID NO:
• 20 and SEQ ID NO:
• 21;
• (b) a second pair of PCR primers comprising SEQ ID NO:
• 22 and SEQ ID NO:
• 23;
• (c) a negative control comprising a wild-type km23 cDNA;
• and
• (d) a positive control comprising a km23 mutant cDNA.