## Kinase co-regulation identifies novel molecular effectors

The conditional depth of the kinase regulation atlas facilitates the search for co-regulated kinases and potential molecular effectors. Protein complexes are common signaling effectors that often display coordinated phospho-regulation with regulatory kinases. To search for kinase-complex co-regulation, we quantified the enrichment of regulated phosphosites within stable human complexes. We then correlate this enrichment with the KSEA activities across the panel of biological perturbations (Materials and Methods). Kinase-complex associations were validated if at least one subunit in the complex was a known substrate of the kinase. Overall, we found a very strong enrichment for known kinase targets among the kinase-complex associations predicted from co-regulation (Fig 5A, Table EV6). Using CDK1 as an example, we found a significant number of co-regulated complexes validated as direct substrates of CDK1 based on previous evidence, even though the actual substrate sites in the complex were not used to predict their association (Fig 5B). We have also identified examples of complex subunits functionally related with CDK1, but with no evidence yet of direct regulation. The chromatin assembly complex (CAF-1 Complex), for instance, delivers newly synthesized H3/H4 dimers to the replication fork during the DNA synthesis (S) phase, shifting to secondary functions during other stages of the cell cycle \cite{26066981}. Although no specific site in the complex has been validated as CDK1 substrate, the observed co-regulation of CAF-1 and CDK1 (\(r=0.27\), \(FDR=5\times10^{-4}\)) was partially validated *in vitro* as CDK inhibition prevents the replication-dependent nucleosome assembly in human cell extracts \cite{10938080}. 

As an additional application of this approach, we tested if co-regulation can also be predictive of novel AKT1 kinase target sites. In order to validate these predictions we measured the phosphorylation changes of 15,255 phosphosites in insulin-stimulated HeLa cells in the presence or absence of the AKT inhibitor VIII (Fig 5C, Table EV7). As expected, previously known AKT targets are, on average, down-regulated in the presence of the inhibitor (Fig 5D). Additionally, the substrates of downstream related kinases, such as MTOR or GSK, are also regulated. When predicting the same number of AKT targets either as sites strongly matching the AKT sequence preference or sites showing the most significant co-regulation with AKT across conditions, the latter showed much stronger down-regulation after AKT inhibition (Fig 5D).

In order to propose a list of new _bona fide_ AKT targets, we shortlisted those that are strongly co-regulated with the AKT activity (\(p \lt 0.01\)), match the AKT sequence preference, are down-regulated after AKT inhibition (\(\log_{2}L/H\lt-0.9\)) and were also reported as *in vitro* AKT substrate sites \cite{24869485}. We identified 12 AKT target phosphosites matching these stringent criteria (Fig 5E, Table EV8). S588 of TBC1D4 was previously described to be a potential AKT target sites, despite it not being present in our training set \cite{12107176,11994271}. The tumor suppressor PDCD4 has also been shown to be regulated by AKT but at position S67 controlling its localization \cite{16357133}. We identified S76 as an AKT target, a residue important for PDCD4 degradation \cite{24334270,25002506} that is phosphorylated under EGF stimulation \cite{24334270}. This suggests that AKT activity may directly control the protein levels of PDCD4. Another interesting target site is S801 of the kinesin KIF4A, a motor protein involved in the control of microtubule stability. The nearby position T799 is a target of Aurora B and the double alanine mutant T799A/S801A has impaired function \cite{23940115}. In addition, both AKT and KIF4A regulate microtubule stability during cell migration \cite{24658398,17397400}. Taken together, these results suggest that AKT directly targets KIF4A S801 and that this interaction plays a role in microtubule stabilization during migration.

