C. Lukas et al., Immunochemical analysis of the D-type cyclin-dependent kinases Cdk4 and Cdk6, using a series of monoclonal antibodies, HYBRIDOMA, 18(3), 1999, pp. 225-234
Cellular signal transduction cascades triggered by mitogenic or antiprolife
rative cues eventually converge on a biochemical mechanism centered around
the retinoblastoma tumor suppressor (pRb): the so-called RE pathway that go
verns G1-phase progression and guards the commitment to enter S phase. pRb,
together with its immediate upstream regulators, the D-type cyclins, their
partner cyclin-dependent kinases Cdk4 and Cdk6, and the Cdk inhibitors, fo
rm a functional unit that is involved in major decisions about cellular fat
e, and whose components, including the proto-oncogenic cyclin D-dependent k
inases, are commonly deregulated in many types of cancer. We report here th
e production and characterization of a series of 12 monoclonal antibodies (
MAbs) that specifically recognize either Cdk4 or Cdk6. These antibodies are
proving to be invaluable molecular probes for defining abundance, subcellu
lar localization, binding partners, and ultimately the function(s) of these
cell cycle-regulatory kinases. Localization of the target epitopes was map
ped by peptide enzyme-linked immunoadsorbent assay (ELISA), and two antibod
ies recognizing sequences adjacent to N-terminus of Cdk4 can discriminate b
etween the wild-type protein and the oncogenic, melanoma-associated R24C mu
tant of this kinase. Individual antibodies of our panel recognize distinct
pools of Cdk4/6, a feature reflected by their differential applicability in
immunoblotting, immunoprecipitation, kinase assays, and immunostaining inc
luding immunohistochemistry an archival paraffin-embedded tissue sections.
Collectively, the antibodies described in this study provide the means for
immunochemical analyses of the cyclin D-dependent kinases in human and anim
al cells, and represent useful molecular tools that should help better unde
rstand the biological roles of Cdk4 and Cdk6 in normal cell-cycle control,
and their oncogenic activity in tumor cells.