Expression of adaptor protein Ruk/CIN85 isoforms in cell lines of various tissue origins and human melanoma.
OBJECTIVE
Development of monoclonal and polyclonal antibodies against recombinant GST-fused proteins including correspondingly N- and C-terminal parts of Ruk/CIN85 adaptor protein. Analysis of Ruk/CIN85 expression patterns in cell lines of various tissue origins and human melanoma.


METHODS
Recombinant GST-fused fragments of Ruk/CIN85 were expressed in bacterial system and affinity purified. Monoclonal antibodies against SH3A domain of Ruk/CIN85 were produced using hybridoma technique. The specificity of generated antibodies was examined by ELISA. Polyclonal antibodies against C-terminal coiled-coil region of Ruk/CIN85 were affinity purified from serum of immunized rabbit. Expression patterns of Ruk/CIN85 isoforms and their subcellular localization in cell lines of various tissue origins and human melanoma samples were analyzed by immunoblotting, immunoprecipitation and immunofluorescence microcopy.


RESULTS
Ruk/CIN85 is ubiquitously expressed SH3-containing adaptor/scaffold protein which plays important roles in signalling processes. N-terminal half of Ruk/CIN85 molecule, including three SH3 domains, and its C-terminal coiled-coil region were used as antigens to produce monoclonal and polyclonal antibodies, respectively. Hybridoma cell lines secreting monoclonal antibodies (mAbs) to SH3 fragment of Ruk/CIN85 were established. One of the mAbs was extensively characterized and designated as MISh-A1. It was shown that this mAb recognizes an epitope, which resides within first SH3A domain. Polyclonal anti-Ruks Abs affinity purified from serum of immunized rabbit specifically recognized main Ruk/CIN85 isoforms, both endogenous and recombinant, in lysates of HEK293 cells. Notably, produced Abs did not cross-react with CD2AP, the member of the same family of adaptor/scaffold proteins. Multiple molecular forms of Ruk/CIN85 with apparent molecular weights of 130, 80-85, 70-75, 50-56, 34-40 and 29 kD were detected in cell lyzates of NIH3T3, Cos1, L1210, HEK293, Ramos, HeLa S3, MDCK, C6, A549 and U937 using anti-Ruk antibodies. Oligomerization between p85 and p50-56 forms of Ruk/CIN85 was revealed in C6 and NIH3T3 cells, but not in HeLa S3 and HEK293 cells by immunoprecipitation using MISh-A1 antibody following anti-Ruk Western-blot analysis. Using immunofluorescent microscopy and anti-Ruk antibodies, endogenous Ruk-variates were found mostly in cytoplasm of C6, NIH3T3, HEK293 cells and at lower level - in nuclei.


CONCLUSIONS
Patterns of Ruk/CIN85 molecular forms expression are cell-specific and determined by cellular context. Assembly of oligomeric complexes between p85 and p50-56 Ruk/CIN85 isoforms in C6 and NIH3T3 cells but not in HeLa S3 and HEK293 cells may reflect their specific biological roles in different cell lines. High level of full-length Rukl/CIN85 form expression was revealed in extracts of human melanoma samples. Abs described in this paper may prove useful in future studies of Ruk/CIN85 expression and function in normal and transformed cells.
Mayevska
O
O
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv 79005, Ukraine.
Shuvayeva
H
H
Igumentseva
N
N
Havrylov
S
S
Basaraba
O
O
Bobak
Y
Y
Barska
M
M
Volod'ko
N
N
Baranska
J
J
Buchman
V
V
Drobot
L
L
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation; D008545:Melanoma; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D011994:Recombinant Proteins; C433810:SH3KBP1 protein, human