TY - JOUR
T1 - CDK6 activity in a recurring convergent kinase network motif
AU - Gangemi, Christina G.
AU - Sabapathy, Rahkesh T.
AU - Janovjak, Harald
PY - 2023/4
Y1 - 2023/4
N2 - In humans, more than 500 kinases phosphorylate ~15% of all proteins in an emerging phosphorylation network. Convergent local interaction motifs, in which ≥two kinases phosphorylate the same substrate, underlie feedback loops and signal amplification events but have not been systematically analyzed. Here, we first report a network-wide computational analysis of convergent kinase-substrate relationships (cKSRs). In experimentally validated phosphorylation sites, we find that cKSRs are common and involve >80% of all human kinases and >24% of all substrates. We show that cKSRs occur over a wide range of stoichiometries, in many instances harnessing co-expressed kinases from family subgroups. We then experimentally demonstrate for the prototypical convergent CDK4/6 kinase pair how multiple inputs phosphorylate the tumor suppressor retinoblastoma protein (RB) and thereby hamper in situ analysis of the individual kinases. We hypothesize that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells expressing high levels of CDK4, we confirm this hypothesis and develop a high-throughput compatible assay that quantifies genetically modified CDK6 variants and inhibitors. Collectively, our work reveals the occurrence, topology, and experimental dissection of convergent interactions toward a deeper understanding of kinase networks and functions.
AB - In humans, more than 500 kinases phosphorylate ~15% of all proteins in an emerging phosphorylation network. Convergent local interaction motifs, in which ≥two kinases phosphorylate the same substrate, underlie feedback loops and signal amplification events but have not been systematically analyzed. Here, we first report a network-wide computational analysis of convergent kinase-substrate relationships (cKSRs). In experimentally validated phosphorylation sites, we find that cKSRs are common and involve >80% of all human kinases and >24% of all substrates. We show that cKSRs occur over a wide range of stoichiometries, in many instances harnessing co-expressed kinases from family subgroups. We then experimentally demonstrate for the prototypical convergent CDK4/6 kinase pair how multiple inputs phosphorylate the tumor suppressor retinoblastoma protein (RB) and thereby hamper in situ analysis of the individual kinases. We hypothesize that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells expressing high levels of CDK4, we confirm this hypothesis and develop a high-throughput compatible assay that quantifies genetically modified CDK6 variants and inhibitors. Collectively, our work reveals the occurrence, topology, and experimental dissection of convergent interactions toward a deeper understanding of kinase networks and functions.
KW - convergence
KW - inhibitor
KW - kinase
KW - protein engineering
KW - signaling network
UR - http://www.scopus.com/inward/record.url?scp=85149999758&partnerID=8YFLogxK
UR - http://purl.org/au-research/grants/ARC/FT200100519
UR - http://purl.org/au-research/grants/ARC/DP200102093
UR - http://purl.org/au-research/grants/NHMRC/1187638
U2 - 10.1096/fj.202201344R
DO - 10.1096/fj.202201344R
M3 - Article
C2 - 36884374
AN - SCOPUS:85149999758
SN - 0892-6638
VL - 37
JO - FASEB journal : official publication of the Federation of American Societies for Experimental Biology
JF - FASEB journal : official publication of the Federation of American Societies for Experimental Biology
IS - 4
M1 - e22845
ER -