We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p6Osrc, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60`sc proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p6oSrc proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60`rc proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60`rc was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p6Osrc proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p6tVrc apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.

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An oligonucleotide labeling system was developed that can produce radiolabeled hybridization probes with tenfold or more higher specific activity than is obtained by traditional 5'-end-labeling with polynucleotide kinase. Yet the system is as rapid and simple as kinase labeling. The reaction uses the Klenow fragment of E. coli DNA polymerase to add alpha-32P-dA residues to the 3'-end of an oligonucleotide in a primer-extension reaction. Unlike other methods of radioactive tailing (e.g., terminal transferase), a single species is produced of both known length and known specific activity. The reaction is efficient, and over 90% of probe molecules are routinely labeled. Using this method of labeling, an oligonucleotide was shown to be tenfold more sensitive in detecting target DNA sequences in a dot blot hybridization assay, compared to the same oligonucleotide labeled using polynucleotide kinase. Northern blots of Schizosaccharomyces pombe RNA were probed with an oligonucleotide specific for intron 1 of the tf2d gene, a TATA-box binding transcription factor. Kinase-labeled tf2d probe detected only unspliced RNA, while the same oligonucleotide labeled using the new method detected both unspliced tf2d RNA and rare pre-mRNA splicing intermediates.

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CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. CK1 kinases have highly similar catalytic domains, plus a conserved extension to that kinase domain that is important for enzyme stability and activity. In contrast to the catalytic domains, the C‐terminal tails of CK1 family members diverge in sequence and length; however, they all appear to serve as substrates of autophosphorylation. The autophosphorylated tails are proposed to inhibit kinase activity by acting as pseudosubstrates. In addition to C‐terminal autophosphorylation, autophosphorylation of the CK1□ kinase domain has been detected, but its effect on CK1 activity and cellular function has never been explored. Here, we addressed the role of kinase domain autophosphorylation in human CK1□ and CK1□, as well as Hhp1 and Hhp2, their homologues in Schizosaccharomyces pombe. In each case, we found that autophosphorylation of a conserved threonine residue in the kinase domain inhibited enzyme activity. This site resides in the mobile L‐EF loop proximal to the active site, distinct from the well‐characterized T‐loop autophosphorylation that occurs in other kinase families. We found that yeast and human enzymes with phosphoablating mutations at this site are hyperactive in vitro. In vivo, mutation of this site protects yeast cells from heat shock, indicating a change in substrate profile, a prediction we are currently testing using quantitative phosphoproteomics. We have also found that autophosphorylation of the kinase domain may affect autophosphorylation of the C‐terminus, so there is likely interplay between the two different modalities of CK1 autoregulation that ultimately determines the extent to which substrates are phosphorylated. We propose that phosphorylation on the L‐EF loop prevents substrate docking with the kinase domain by shielding the positively charged substrate binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L‐EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo. Support or Funding Information This work was supported by R35‐GM131799 (to KLG) and T32‐CA119925 (to SNC).

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CK1 enzymes signal in a variety of important cellular pathways, including DNA damage repair, mitotic checkpoint signaling, circadian rhythm, Wnt signaling, endocytosis, and neurodegenerative disease progression. Like other multifunctional kinases, CK1 must be regulated in space and time in order to target specific subsets of its substrates in each of the pathways it participates in. However, CK1 is generally regarded as a “rogue” kinase, which is constitutively active, ubiquitous throughout cells and tissues, and unregulated except by autoinhibition.CK1 enzymes are known to autophosphorylate their C‐terminal non‐catalytic tails, which are proposed to inhibit their activity by acting as pseudosubstrates. This model would require a phosphorylation‐dependent intramolecular interaction between the C‐terminus and the kinase domain, but we are unaware of any evidence demonstrating such an event. Furthermore, this proposed mechanism of autoinhibition has not been tested in vivo in any organism.We have identified six serine and threonine autophosphorylation sites on the C‐terminus of Schizosaccharomyces pombe Hhp1, one of two soluble CK1 enzymes in this organism, and are testing candidate sites on Hhp2 and the human homologues CK1□/□. When these sites are specifically phosphorylated, the Hhp1 C‐terminus binds the kinase domain via a low‐affinity electrostatic interaction. At concentrations above the Kd of this interaction, the phosphorylated C‐terminus inhibits Hhp1 kinase activity, while mutations that abolish phosphorylation increase the activity of the full‐length kinase.Structural studies have identified two conserved basic patches on the CK1 kinase domain that are hypothesized to interact with the phosphorylated C‐terminus; however, we show that the tail instead interacts with the substrate binding pocket. The physiological substrate Sid4 has a higher affinity than the tail for the Hhp1 kinase domain, and the model substrate casein can out‐compete the phosphorylated C‐terminus for binding to the kinase domain. Our data support a new model of CK1 activation in which the presence of high‐affinity substrates is sufficient to displace the low‐affinity tail and relieve autoinhibition without the need for prior dephosphorylation of CK1.Because the C‐termini of CK1 family members are responsible for most of the sequence variation between isoforms, we hypothesize that these different tails may differ in affinity for the conserved kinase domain, leading to displacement by different cohorts of substrates. Thus, this mechanism may also explain variations in substrate specificities between CK1 enzymes. In support of this model, truncating the tail of Hhp1 or Hhp2 eliminates substrate‐specific differences in catalytic efficiency. We are currently using phosphorylation site mutants of hhp1 to confirm that these changes in catalytic efficiency are dependent on autophosphorylation and to investigate the significance of autophosphorylation in vivo.Support or Funding InformationSNC was supported by the Integrated Biological Systems Training in Oncology Program (2T32CA119925). This work was supported by NIH GM112989 to KLG.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.