Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

ABLs and TMKs are co-receptors for extracellular auxin.

Extracellular perception of auxin, an essential phytohormone in plants, has been debated for decades. Auxin-binding protein 1 (ABP1) physically interacts with quintessential transmembrane kinases (TMKs) and was proposed to act as an extracellular auxin receptor, but its role was disputed because abp1 knockout mutants lack obvious morphological phenotypes. Here, we identified two new auxin-binding proteins, ABL1 and ABL2, that are localized to the apoplast and directly interact with the extracellular domain of TMKs in an auxin-dependent manner. Furthermore, functionally redundant ABL1 and ABL2 genetically interact with TMKs and exhibit functions that overlap with those of ABP1 as well as being independent of ABP1. Importantly, the extracellular domain of TMK1 itself binds auxin and synergizes with either ABP1 or ABL1 in auxin binding. Thus, our findings discovered auxin receptors ABL1 and ABL2 having functions overlapping with but distinct from ABP1 and acting together with TMKs as co-receptors for extracellular auxin.

Mechanisms for Regulating and Organizing Receptor Signaling by Endocytosis.

Intricate relationships between endocytosis and cellular signaling, first recognized nearly 40 years ago through the study of tyrosine kinase growth factor receptors, are now known to exist for multiple receptor classes and to affect myriad physiological and developmental processes. This review summarizes our present understanding of how endocytosis orchestrates cellular signaling networks, with an emphasis on mechanistic underpinnings and focusing on two receptor classes-tyrosine kinase and G protein-coupled receptors-that have been investigated in particular detail. Together, these examples provide a useful survey of the current consensus, uncertainties, and controversies in this rapidly advancing area of cell biology.

Checkpoint Responses to DNA Double-Strand Breaks.

Cells confront DNA damage in every cell cycle. Among the most deleterious types of DNA damage are DNA double-strand breaks (DSBs), which can cause cell lethality if unrepaired or cancers if improperly repaired. In response to DNA DSBs, cells activate a complex DNA damage checkpoint (DDC) response that arrests the cell cycle, reprograms gene expression, and mobilizes DNA repair factors to prevent the inheritance of unrepaired and broken chromosomes. Here we examine the DDC, induced by DNA DSBs, in the budding yeast model system and in mammals.

Calcium Signaling Mechanisms Across Kingdoms.

Calcium (Ca2+) is a unique mineral that serves as both a nutrient and a signal in all eukaryotes. To maintain Ca2+ homeostasis for both nutrition and signaling purposes, the tool kit for Ca2+ transport has expanded across kingdoms of eukaryotes to encode specific Ca2+ signals referred to as Ca2+ signatures. In parallel, a large array of Ca2+-binding proteins has evolved as specific sensors to decode Ca2+ signatures. By comparing these coding and decoding mechanisms in fungi, animals, and plants, both unified and divergent themes have emerged, and the underlying complexity will challenge researchers for years to come. Considering the scale and breadth of the subject, instead of a literature survey, in this review we focus on a conceptual framework that aims to introduce readers to the principles and mechanisms of Ca2+ signaling. We finish with several examples of Ca2+-signaling pathways, including polarized cell growth, immunity and symbiosis, and systemic signaling, to piece together specific coding and decoding mechanisms in plants versus animals.

Processing Temporal Growth Factor Patterns by an Epidermal Growth Factor Receptor Network Dynamically Established in Space.

The proto-oncogenic epidermal growth factor (EGF) receptor (EGFR) is a tyrosine kinase whose sensitivity and response to growth factor signals that vary over time and space determine cellular behavior within a developing tissue. The molecular reorganization of the receptors on the plasma membrane and the enzyme-kinetic mechanisms of phosphorylation are key determinants that couple growth factor binding to EGFR signaling. To enable signal initiation and termination while simultaneously accounting for suppression of aberrant signaling, a coordinated coupling of EGFR kinase and protein tyrosine phosphatase activity is established through space by vesicular dynamics. The dynamical operation mode of this network enables not only time-varying growth factor sensing but also adaptation of the response depending on cellular context. By connecting spatially coupled enzymatic kinase/phosphatase processes and the corresponding dynamical systems description of the EGFR network, we elaborate on the general principles necessary for processing complex growth factor signals.

Structural and Functional Analysis of a ß2-Adrenergic Receptor Complex with GRK5.

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.

Trafficking of Adhesion and Growth Factor Receptors and Their Effector Kinases.

Cell adhesion to macromolecules in the microenvironment is essential for the development and maintenance of tissues, and its dysregulation can lead to a range of disease states, including inflammation, fibrosis, and cancer. The biomechanical and biochemical mechanisms that mediate cell adhesion rely on signaling by a range of effector proteins, including kinases and associated scaffolding proteins. The intracellular trafficking of these must be tightly controlled in space and time to enable effective cell adhesion and microenvironmental sensing and to integrate cell adhesion with, and compartmentalize it from, other cellular processes, such as gene transcription, protein degradation, and cell division. Delivery of adhesion receptors and signaling proteins from the plasma membrane to unanticipated subcellular locales is revealing novel biological functions. Here, we review the expected and unexpected trafficking, and sites of activity, of adhesion and growth factor receptors and intracellular kinase partners as we begin to appreciate the complexity and diversity of their spatial regulation.

LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation.

Deregulated inflammation is a critical feature driving the progression of tumors harboring mutations in the liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB-regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt-inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.

The multiple roles of salt-inducible kinases in regulating physiology.

Salt-inducible kinases (SIKs), which comprise a family of three homologous serine-threonine kinases, were first described for their role in sodium sensing but have since been shown to regulate multiple aspects of physiology. These kinases are activated or deactivated in response to extracellular signals that are cell surface receptor mediated and go on to phosphorylate multiple targets including the transcription cofactors CRTC1-3 and the class IIa histone deacetylases (HDACs). Thus, the SIK family conveys signals about the cellular environment to reprogram transcriptional and posttranscriptional processes in response. In this manner, SIKs have been shown to regulate metabolic responses to feeding/fasting, cell division and oncogenesis, inflammation, immune responses, and most recently, sleep and circadian rhythms. Sleep and circadian rhythms are master regulators of physiology and are exquisitely sensitive to regulation by environmental light and physiological signals such as the need for sleep. Salt-inducible kinases have been shown to be central to the molecular regulation of both these processes. Here, we summarize the molecular mechanisms by which SIKs control these different domains of physiology and highlight where there is mechanistic overlap with sleep/circadian rhythm control.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation.

NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.

RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer.

Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.

Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation.

Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.

Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication.

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.

Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.

Rewiring of the phosphoproteome executes two meiotic divisions in budding yeast.

The cell cycle is ordered by a controlled network of kinases and phosphatases. To generate gametes via meiosis, two distinct and sequential chromosome segregation events occur without an intervening S phase. How canonical cell cycle controls are modified for meiosis is not well understood. Here, using highly synchronous budding yeast populations, we reveal how the global proteome and phosphoproteome change during the meiotic divisions. While protein abundance changes are limited to key cell cycle regulators, dynamic phosphorylation changes are pervasive. Our data indicate that two waves of cyclin-dependent kinase (Cdc28Cdk1) and Polo (Cdc5Polo) kinase activity drive successive meiotic divisions. These two distinct phases of phosphorylation are ensured by the meiosis-specific Spo13 protein, which rewires the phosphoproteome. Spo13 binds to Cdc5Polo to promote phosphorylation in meiosis I, particularly of substrates containing a variant of the canonical Cdc5Polo motif. Overall, our findings reveal that a master regulator of meiosis directs the activity of a kinase to change the phosphorylation landscape and elicit a developmental cascade.