Neil J. Grimsey

Protein phosphorylation is a fundamental mechanism regulating signal transduction, metabolic control, and cellular adaptation to environmental stress, with these processes orchestrated largely by protein kinases organized into hierarchical signaling cascades. Among these, the p38 mitogen-activated protein kinases (MAPKs) play a central role in stress and inflammatory signaling and are classically characterized by activation by upstream kinases MKK3 and MKK6. However, accumulating biochemical, structural, and cellular evidence demonstrates that p38—particularly the p38α isoform—can also be activated through atypical, MKK3/6-independent mechanisms driven by direct protein–protein interactions that promote kinase autophosphorylation. In the present review, we synthesize current insights into atypical p38 activation, comparing evidence for signaling mediated by the transforming growth factor β-activated kinase 1 binding protein 1, zeta-chain-associated protein kinase 70, the Hepatitis C virus core protein, and the Toxoplasma gondii protein GRA24. We examine the molecular and structural determinants of their interactions, the conformational changes that enable cis-autophosphorylation, and the resulting differences in regulation and substrate engagement compared with canonical p38 signaling. We further discuss evidence linking atypical p38 activation to selective pathological outcomes in models of cardiac ischemia, infection, inflammation, and vascular dysfunction. Together, these studies establish atypical p38 signaling as a distinct biochemical regulatory paradigm that expands our understanding of MAPK signaling specificity and context-dependent kinase function.

Despite the life‐changing impact of anti‐VEGF therapies, vascular retinopathies continue to affect millions of patients worldwide. To better understand disease progression, it is essential to define alternative mechanisms that contribute to retinal vascular dysregulation. Mitogen‐activated protein kinase (MAPK) p38 plays a significant role in regulating vascular homeostasis, angiogenesis, and retinal disease progression, yet effective therapeutic targeting remains elusive. An alternative atypical p38 signaling pathway is mediated by interaction with the adaptor protein TGF‐beta activated kinase 1, binding protein 1 (TAB1). Atypical p38 can be activated by ischemia or by inflammatory G protein‐coupled receptors (GPCRs) to regulate inflammation and vascular homeostasis. However, atypical signaling has not been investigated in the context of vascular retinopathies, specifically oxygen‐induced retinopathy (OIR). Here, we utilized a genetic knock‐in mouse to block atypical p38 activity (Tab1 ^KI ) to explore retinal damage during OIR. We report that Tab1 ^KI mice display significantly reduced vaso‐obliteration and limited neovascularization relative to wild‐type C57BL6 controls. Retinal RNAseq analysis revealed distinct transcriptional regulation in the Tab1 ^KI mouse. Suppression of atypical p38 reduced Mef2c signaling, which in turn enhanced microglial activation and inflammation. Despite the upregulation of proangiogenic markers, the increase in endothelial markers does not correlate with the dysregulated growth of blood vessels. Instead, Mef2c suppression reduces pathological angiogenesis while increasing physiological vascular regrowth. The combined data suggest that the selective inhibition of atypical p38 signaling could block the enhanced pathogenic neovascular tuft formation without impacting blood vessel repair. Further investigation will clarify how atypical p38 controls neovascular responses, potentially revealing new treatment strategies for vascular retinopathies.

Developing therapeutics that target the host kinome is a promising strategy to support current treatments for viral infections. While multiple direct‐acting antivirals (DAAs) potentially clear Hepatitis C virus (HCV) infection in most individuals, ~2.4 million individuals are unresponsive to the current DAAs or have secondary infections/complications that impact their treatment potential. Recent studies have demonstrated that atypical p38 activity is crucial for HCV replication and may have broader implications in regulating other viruses. Despite the multifaceted roles of p38 in regulating viral pathogenesis, the molecular mechanisms that underlie atypical p38 activation by viruses remain a critical gap in our understanding of viral physiology. Here, we characterized two atypical p38 allosteric modulators (NC compounds) during HCV infection. We compared the effects of these compounds to current DAAs for HCV and Herpesvirus, respectively. Our data show that treatment of either NC compound rapidly attenuates HCV‐induced p38 activation, blocking HCV protein expression and HCV RNA replication. Interestingly, while both NC compounds can successfully attenuate HCV infection, we also found that they displayed the capacity to inhibit the betaherpesvirus, Human Cytomegalovirus (HCMV), and the alpha herpesvirus, Herpes Simplex Virus‐1 (HSV‐1). Our study is the first to assess the potential of atypical p38 selective modulators to block viral replication. While the current examples are likely not clinically viable, the concept represents an exciting opportunity to support or complement current treatment strategies, shedding light on the importance that host factors play during viral physiology.

Mitogen‐activated protein kinase (MAPK) p38 plays a key role in driving the pathology of acute lung injury (ALI), but effective therapeutic targeting remains elusive. Atypical p38 signaling, mediated by interaction with the adaptor protein Tumor Growth Factor β Activated Kinase 1 (TAK1) Binding Protein 1 (TAB1), has so far only been observed during pathological responses, representing a selective and alternative target during pulmonary injury. However, atypical signaling has not been investigated in the context of pulmonary injury and immune responses related to the onset and progression of ALI. Here, we utilized a genetic knock‐in mouse to block influenza A‐induced lung injury mediated by atypical signaling. We report that the loss of TAB1‐p38 interaction reduces weight loss and recovery time, reduces histopathological scores associated with influenza‐induced lung injury early during infection, and prompts earlier recruitment of monocytes to the lungs following infection. These results were found to be independent of viral replication and infectivity, representing the first evidence for the roles of atypical signaling as a driver of host‐mediated pulmonary injury following influenza infection.

Despite the life-changing impact of anti-VEGF therapies, vascular retinopathies continue to affect millions of patients worldwide. To better understand disease progression, it is essential to define alternative mechanisms that contribute to retinal vascular dysregulation. Mitogen-activated protein kinase (MAPK) p38 plays a significant role in regulating vascular homeostasis, angiogenesis, and retinal disease progression, yet effective therapeutic targeting remains elusive. An alternative atypical p38 signaling pathway is mediated by interaction with the adaptor protein TGF-beta activated kinase 1, binding protein 1 (TAB1). Atypical p38 can be activated by ischemia or by inflammatory G protein-coupled receptors (GPCRs) to regulate inflammation and vascular homeostasis. However, atypical signaling has not been investigated in the context of vascular retinopathies, specifically oxygen-induced retinopathy (OIR). Here, we utilized a genetic knock-in mouse to block atypical p38 activity (Tab1 ^KI ) to explore retinal damage during OIR. We report that Tab1 ^KI mice display significantly reduced vaso-obliteration and limited neovascularization relative to wild-type C57BL6 controls. Retinal RNAseq analysis revealed distinct transcriptional regulation in the Tab1 ^KI mouse. Suppression of atypical p38 reduced Mef2c signaling, which in turn enhanced microglial activation and inflammation. Despite the upregulation of proangiogenic markers, the increase in endothelial markers does not correlate with the dysregulated growth of blood vessels. Instead, Mef2c suppression reduces pathological angiogenesis, while increasing physiological vascular regrowth. The combined data suggest that the selective inhibition of atypical p38 signaling could block the enhanced pathogenic neovascular tuft formation without impacting blood vessel repair. Further investigation will clarify how atypical p38 controls neovascular responses, potentially revealing new treatment strategies for vascular retinopathies. NEW AND NOTEWORTHY: MAPK p38 drives ocular injury and inflammation, but the contribution of Tab1-dependent atypical p38 in retinal injury has not been investigated. Using a mouse genetic knock-in model to block atypical p38 during oxygen-induced retinal injury, we show that atypical p38 enhances vascular damage and neovascular tufting. Blockade of atypical p38 protects mice from retinal vascular damage.

Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 is a significant contributor to suppression of NOTCH1 activation by JAG1 or enhancement of NOTCH1 activation by DLL1, respectively. In this study we sought to evaluate the significance of these glycosylation sites in a mammalian model by generating two C57BL/6 J mouse lines carrying NOTCH1 point mutations which eliminate O-fucosylation and Fringe activity at EGFs 6 (T232V) or 8 (T311V). We assessed changes to morphology during retinal angiogenesis, a process in which expression of Notch1, Jag1, Dll4, Lfng, Mfng, and Rfng genes coordinate cell-fate decisions to grow vessel networks. In the EGF6 O-fucose mutant (6f/6f) retinas we observed reduced vessel density and branching, suggesting that this mutant is a Notch1 hypermorph. This finding agrees with prior cell-based studies showing that the 6f mutation increased JAG1 activation of NOTCH1 during co-expression with inhibitory Fringes. Although we predicted that the EGF8 O-fucose mutant (8f/8f) would not complete embryonic development due to the direct involvement of the O-fucose in engaging ligand, the 8f/8f mice were viable and fertile. In the 8f/8f retina we measured increased vessel density consistent with established Notch1 hypomorphs. Overall, our data supports the importance of NOTCH1 O-fucose residues for pathway function and confirms that single O-glycan sites are rich in signaling instructions for mammalian development.

Mitogen-activated protein kinase (MAPK) p38 is a central regulator of intracellular signaling, driving physiological and pathological pathways. With over 150 downstream targets, it is predicted that spatial positioning and the availability of cofactors and substrates determines kinase signaling specificity. The subcellular localization of p38 is highly dynamic to facilitate the selective activation of spatially restricted substrates. However, the spatial dynamics of atypical p38 inflammatory signaling are understudied. We utilized subcellular targeted fluorescence resonance energy transfer (FRET) p38 activity biosensors to map the spatial profile of kinase activity. Through comparative analysis of plasma membrane, cytosolic, nuclear, and endosomal compartments, we confirm a characteristic profile of nuclear bias for mitogen-activated kinase kinase 3/6 (MKK3/6) dependent p38 activation. Conversely, atypical p38 activation via thrombin-mediated protease-activated receptor 1 (PAR1) activity led to enhanced p38 activity at the endosome and cytosol, limiting nuclear p38 activity, a profile conserved for prostaglandin E2 activation of p38. Conversely, perturbation of receptor endocytosis led to spatiotemporal switching of thrombin signaling, reducing endosomal and cytosolic p38 activity and increasing nuclear activity. The data presented reveal the spatiotemporal dynamics of p38 activity and provide critical insight into how atypical p38 signaling drives differential signaling responses through spatial sequestration of kinase activity.

Mitogen-activated protein kinase (MAPK) p38 is a central regulator of intracellular signaling, driving physiological and pathological pathways. With over 150 downstream targets, it is predicted that spatial positioning and the availability of cofactors and substrates determines kinase signaling specificity. The subcellular localization of p38 is highly dynamic to facilitate the selective activation of spatially restricted substrates. However, the spatial dynamics of atypical p38 inflammatory signaling are understudied. We developed spatially targeted fluorescence resonance energy transfer (FRET) biosensors to track p38 activity with subcellular resolution. Through comparative analysis of plasma membrane, cytosolic, nuclear, and endosomal compartments, we confirm a characteristic profile of nuclear bias for mitogen-activated kinase kinase 3/6 (MKK3/6) dependent p38 activation. Conversely, atypical p38 activation via thrombin-mediated protease-activated receptor 1 (PAR1) activity led to the sequestration of p38 at the endosome and cytosol, limiting nuclear translocation, a profile conserved for prostaglandin E2 activation of p38. Conversely, perturbation of receptor endocytosis led to spatiotemporal switching of thrombin signaling, reducing endosomal and cytosolic p38 activation and increasing nuclear activity. The data presented reveal the spatiotemporal dynamics of p38 activity and provide critical insight into how atypical p38 signaling drives differential signaling responses through spatial sequestration of kinase activity.