Potential coupling between SARS-CoV-2 replicative fitness and interactions of its nucleoprotein with human 14-3-3 proteins

The SARS-CoV-2 nucleocapsid protein (N) is responsible for viral genome packaging and virion assembly. Being highly abundant in the host cell, N interacts with numerous human proteins and undergoes multisite phosphorylation in vivo. When phosphorylated within its Ser/Arg-rich region, a tract highly prone to mutations as exemplified in the Omicron and Delta variants, N recruits human 14-3-3 proteins, potentially hijacking their functions. Here, we show that in addition to phosphorylated Ser197, an alternative, less conserved phosphosite at Thr205 not found in SARS-CoV N binds 14-3-3 with micromolar affinity and is in fact preferred over pS197. Fluorescence anisotropy reveals a distinctive pT205/pS197 binding selectivity towards the seven human 14-3-3 isoforms. Crystal structures explain the structural basis for the discovered selectivity towards SARS-CoV-2 N phosphopeptides, and also enable prediction for how N variants interact with 14-3-3, suggesting a link between the strength of this interaction and replicative fitness of emerging coronavirus variants.

ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1

There is considerable evidence that cross-talk between the Hedgehog pathway and MAPK signaling pathways occurs in several types of cancer, and contributes to the emergence of clinical resistance to Hedgehog pathway inhibitors. Here, we demonstrate that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. We show that ERK2 phosphorylates GLI1 on three evolutionarily-conserved target sites (S102, S116 and S130) located near the high-affinity binding site for the negative regulator SUFU; furthermore, these phosphorylation events cooperate to weaken the affinity of GLI1-SUFU binding by over 25 fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily-conserved SUFU-affinity-modulating region. In cultured mammalian cells, mutant GLI1 variants containing phosphomimetic substitutions of S102, S116 and S130 displayed an increased ability to drive transcription. We conclude that of multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

An intrinsic temporal order of c-Jun N-terminal phosphorylation regulates its activity by orchestrating co-factor recruitment

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-Jun proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-Jun multisite phosphorylation has remained elusive. Here we show that c-Jun phosphorylation by JNK exhibits a defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identified the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis revealed three c-Jun phosphorylation states: unphosphorylated c-Jun recruits the Mbd3 repressor, serine63/73 doubly-phosphorylated c-Jun binds to the Tcf4 co-activator, whereas the fully phosphorylated form disfavours Tcf4 binding attenuating JNK signalling. Thus, c-Jun phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.

Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14 and Sic1 for origin licensing in S. cerevisiae

Cdc6, a subunit of the pre-replicative complex, contains multiple regulatory Cdk1 consensus sites, SP or TP motifs. In S. cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S-phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the Cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, the Cdk1 inhibitor, Sic1, releases Clb2-Cdk1-Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing is promoted by the attenuation of distinct CDK-dependent Cdc6 inhibitory mechanisms.

Structural basis of phosphorylation kinetics in the transcriptional activation domain (TAD) of c-Jun and insight of two new phosphorylation sites Ser58 and Thr62

Protein phosphorylation is one of the most important posttranslational modifications observed on biomolecules. Nearly one-third of the cell cycle protein undergoes phosphorylation at some stage of the lifespan. Multi-site phosphorylation is well known in biological systems, including those in transcription factors. Multisite phosphorylation on transcription factors brings about their activation and/or inactivation. c-Jun is one of such transcription factors, whose function is dependent upon the state of phosphorylation. N-terminal phosphorylation required for c-Jun activity, while C-terminal one suppresses its activity. c-Jun contains a transcriptional activation domain (TAD) at N-terminus. It is known that four residues viz., Ser63, Ser73, Thr91 and Thr93 get phosphorylated which is required for its functional dimerization. However, there is no evidence if there exists any phosphorylation kinetics in c-Jun. In this paper, for the first time, it has been demonstrated that there exist phosphorylation kinetics within TAD. NMR based analysis suggested that Ser63 follows the fast kinetic while, Thr91 slow and Ser73 and Thr93 fall in the intermediate category. The four sites follow the following trend in their kinetics Ser63 > Ser73 > Thr93 > Thr91. Similar phosphorylation kinetics was also observed inside the C3H 10T0.5 fibroblast. NMR-based experiments also suggested the phosphorylation of two additional sites at Ser58 and Thr62. However, a detailed significance of these phosphorylation kinetic, as well as newly identified sites, is yet to be discovered.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Ordered phosphorylation of cyclin-dependent kinase (CDK) substrates leads to the sequential transcriptional activation and inhibition of hundreds of cell cycle-regulated genes. We find that Ndd1, an activator of genes required for mitotic progression, is both positively and negatively regulated by CDK activity. CDK activity initially stimulates Ndd1-dependent transcription as cells enter mitosis, but prolonged high CDK activity in a mitotic arrest inhibits transcription. The result is a time-delayed negative feedback circuit that generates a pulse of mitotic gene expression. Our results suggest that high CDK activity catalyzes the formation of multiple weak phosphodegrons on Ndd1, leading to its destabilization. Cyclin specificity and phosphorylation kinetics contribute to the timing of Ndd1 destruction. Failure to degrade Ndd1 in a mitotic arrest leads to elevated mitotic gene expression. We conclude that a combination of positive and negative Ndd1 regulation by CDKs governs the timing and magnitude of the mitotic transcriptional program.

AMPK-dependent phosphorylation is required for transcriptional activation of TFEB/TFE3

Increased autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK activation and mTORC1 inhibition to maintain energy homeostasis and cell survival. TFEB and TFE3 are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by mTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB/TFE3 transcriptional activity upon nutrient starvation, FLCN depletion and pharmacological manipulation of mTORC1 or AMPK. AMPK loss does not affect TFEB/TFE3 nuclear localization nor protein levels but reduces their transcriptional activity. Collectively, we show that mTORC1 specifically controls TFEB/TFE3 cytosolic retention whereas AMPK is essential for TFEB/TFE3 transcriptional activity. This dual and opposing regulation of TFEB/TFE3 by mTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1. Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466/467/469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB/TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance.