Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

Antiviral therapeutics against SARS-CoV-2 are needed to treat the pandemic disease COVID-19. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. Here, we used a genome-wide loss of function CRISPR/Cas9 screen in human lung epithelial cells to identify potential host therapeutic targets. Validation of our screening hits revealed that the kinase SRPK1, together with the closely related SRPK2, were jointly essential for SARS-CoV-2 replication; inhibition of SRPK1/2 with small molecules led to a dramatic decrease (more than 100,000-fold) in SARS-CoV-2 virus production in immortalized and primary human lung cells. Subsequent biochemical studies revealed that SPRK1/2 phosphorylate the viral nucleocapsid (N) protein at sites highly conserved across human coronaviruses and, due to this conservation, even a distantly related coronavirus was highly sensitive to an SPRK1/2 inhibitor. Together, these data suggest that SRPK1/2-targeted therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling response of tissues and cell lines to hormones, growth factors, targeted inhibitors, and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.

The nhr-67 nuclear receptor gene of Caenorhabditis elegans encodes the ortholog of the Drosophila tailless and vertebrate Tlx genes. In C. elegans, nhr-67 plays multiple roles in the development of the uterus during L2 and L3 larval stages. Four pre-VU cells are born in the L2 stage and form the precursor complement for the ventral surface of the mature uterus. One of the four pre-VU cells becomes the anchor cell (AC), which exits the cell cycle and differentiates, while the remaining three VU cells serve as stem cells that populate the ventral uterus. The nhr-67 gene functions in the development of both VU cell lineages and AC differentiation. Hypomorphic mutations in nhr-67 identify a 276bp region of the distal promoter that is sufficient to activate nhr-67 expression in pre-VU cells and the AC. The 276bp region includes 8 conserved potential cis-acting sites, including two E boxes and a nuclear receptor binding site. Mutational analysis demonstrates that the two E boxes are required for expression of nhr-67 in uterine precursor cells. The E/daughterless ortholog HLH-2 binds these sites as a homodimer, thus playing a central role in activating nhr-67 expression in the uterine precursors. At least two other binding activities, one of which may be the nhr-25/Ftz-F1 nuclear receptor transcription factor, also contribute to uterine precursor cell expression. The organization of the nhr-67 uterine precursor enhancer is compared to similar conserved enhancers in the egl-43, lag-2, and lin-3 genes, which contain the same HLH-2-binding E boxes and are similarly expressed in both pre-VU cells and the AC. This basic regulatory module allows the coordinated expression of at least four genes. Expression of genes in different cells that must coordinate to form a mature organ is driven by a shared set of promoter elements, which integrate multiple transcription factor inputs.