The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4) includes the regulatory PSTAIRE helix.
BACKGROUND
The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK) CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4.


RESULTS
To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity.


CONCLUSIONS
Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.
Fraedrich
Kirsten
K
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany. kirsten.fraedrich@viro.med.uni-erlangen.de
Müller
Birthe
B
Grassmann
Ralph
R
D001665:Binding Sites; D056742:Cyclin D2; D051358:Cyclin-Dependent Kinase 4 / Q000737:chemistry / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D016356:Gene Products, tax / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017433:Protein Structure, Secondary; D020798:Two-Hybrid System Techniques
C080336:CCND2 protein, human; D056742:Cyclin D2; D016213:Cyclins; D016356:Gene Products, tax; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4