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Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein–coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme’s activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen–deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein–coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen–deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

The with no lysine (K) (WNK) family of enzymes is best known for control of blood pressure through regulation of the function and membrane localization of ion cotransporters. In mice, global as well as endothelial-specific WNK1 gene disruption results in embryonic lethality due to angiogenic and cardiovascular defects. WNK1 ⁻/⁻ embryos can be rescued by endothelial-specific expression of a constitutively active form of the WNK1 substrate protein kinase OSR1 (oxidative stress responsive 1). Using human umbilical vein endothelial cells (HUVECs), we explored mechanisms underlying the requirement of WNK1–OSR1 signaling for vascular development. WNK1 is required for cord formation in HUVECs, but the actions of the two major WNK1 effectors, OSR1 and its close relative SPAK (STE20/SPS1-related proline-, alanine-rich kinase), are distinct. SPAK is important for endothelial cell proliferation, whereas OSR1 is required for HUVEC chemotaxis and invasion. We also identified the zinc-finger transcription factor Slug in WNK1-mediated control of endothelial functions. Our study identifies a separation of functions for the WNK1-activated protein kinases OSR1 and SPAK in mediating proliferation, invasion, and gene expression in endothelial cells and an unanticipated link between WNK1 and Slug that is important for angiogenesis. Significance With no lysine (K) (WNK)1, which is mutated in pseudohypoaldosteronism type II (PHAII) autosomal dominant hypertension, is a large, complex enzyme essential for development, blood pressure control, and many cellular functions. WNK1 signaling is largely mediated by two downstream protein kinases, OSR1 (oxidative stress responsive 1) and SPAK (STE20/SPS1-related proline-, alanine-rich kinase), sometimes considered redundant in terms of WNK1 function. This study characterizes an essential contribution of WNK1 in angiogenesis and presents a mechanism of clear bifurcation in WNK1-dependent functions between OSR1 and SPAK, with SPAK regulating WNK1 effects on proliferation and OSR1 mediating effects on invasion. Our work also identifies a previously unidentified link between WNK1 and the zinc-finger transcription factor Slug, with implications in cancer biology. This study also suggests potential mechanisms for cardiovascular defects associated with PHAII.

Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.

The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β.

Class I PI3-kinases signal downstream of receptor tyrosine kinases and G protein-coupled receptors and have been implicated in tumorigenesis. Although the oncogenic potential of the PI3-kinase subunit p110α requires its mutational activation, other p110 isoforms can induce transformation when overexpressed in the wild-type state. In wild-type p110α, N345 in the C2 domain forms hydrogen bonds with D560 and N564 in the inter-SH2 (iSH2) domain of p85, and mutations of p110α or p85 that disrupt this interface lead to increased basal activity and transformation. Sequence analysis reveals that N345 in p110α aligns with K342 in p110β. This difference makes wild-type p110β analogous to a previously described oncogenic mutant, p110α-N345K. We now show that p110β is inhibited by p85 to a lesser extent than p110α and is not differentially inhibited by wild-type p85 versus p85 mutants that disrupt the C2-iSH2 domain interface. Similar results were seen in soft agar and focus-formation assays, where p110β was similar to p110α-N345K in transforming potential. Inhibition of p110β by p85 was enhanced by a K342N mutation in p110β, which led to decreased activity in vitro, decreased basal Akt and ribosomal protein S6 kinase (S6K1) activation, and decreased transformation in NIH 3T3 cells. Moreover, unlike wild-type p110β, p110β-K342N was differentially regulated by wild-type and mutant p85, suggesting that the inhibitory C2-iSH2 interface is functional in this mutant. This study shows that the enhanced transforming potential of p110β is the result of its decreased inhibition by p85, due to the disruption of an inhibitory C2-iSH2 domain interface.

Cytoskeletal structure and its regulation are essential for maintenance of the differentiated state of specific types of cells and their adaptation to physiologic and pathophysiologic conditions. Renal glomerular capillaries, composed of podocytes, endothelial cells, and the glomerular basement membrane, have distinct structural and biophysical properties and are the site of injury in many glomerular diseases. Calcineurin inhibitors, immunosuppressant drugs used for organ transplantation and auto-immune diseases, can protect podocytes and glomerular capillaries from injury by preserving podocyte cytoskeletal structure. These drugs cause complications including hypertension and hyperkalemia which are mediated by WNK (With No Lysine) kinases as well as vasculopathy with glomerulopathy. WNK kinases and their target kinases oxidative stress-responsive kinase 1 (OSR1) and SPS1-related proline/alanine-rich kinase (SPAK) have fundamental roles in angiogenesis and are activated by calcineurin inhibitors, but the actions of these agents on kidney vasculature, and glomerular capillaries are not fully understood. We investigated WNK1 expression in cultured podocytes and isolated mouse glomerular capillaries to determine if WNK1 contributes to calcineurin inhibitor-induced preservation of podocyte and glomerular structure. WNK1 and OSR1/SPAK are expressed in podocytes, and in a pattern similar to podocyte synaptopodin in glomerular capillaries. Calcineurin inhibitors increased active OSR1/SPAK in glomerular capillaries, the Young’s modulus (E) of glomeruli, and the F/G actin ratio, effects all blocked by WNK inhibition. In glomeruli, WNK inhibition caused reduced and irregular synaptopodin-staining, abnormal capillary and foot process structures, and increased deformability. In cultured podocytes, FK506 activated OSR1/SPAK, increased lamellipodia, accelerated cell migration, and promoted traction force. These actions of FK506 were reduced by depletion of WNK1. Collectively, these results demonstrate the importance of WNK1 in regulation of the podocyte actin cytoskeleton, biophysical properties of glomerular capillaries, and slit diaphragm structure, all of which are essential to normal kidney function.

Class I PI3-kinases signal downstream of receptor tyrosine kinases and G protein-coupled receptors and have been implicated in tumorigenesis. Although the oncogenic potential of the PI3-kinase subunit p110α requires its mutational activation, other p110 isoforms can induce transformation when overexpressed in the wild-type state. In wild-type p110α, N345 in the C2 domain forms hydrogen bonds with D560 and N564 in the inter-SH2 (iSH2) domain of p85, and mutations of p110α or p85 that disrupt this interface lead to increased basal activity and transformation. Sequence analysis reveals that N345 in p110α aligns with K342 in p110β. This difference makes wild-type p110β analogous to a previously described oncogenic mutant, p110α-N345K. We now show that p110β is inhibited by p85 to a lesser extent than p110α and is not differentially inhibited by wild-type p85 versus p85 mutants that disrupt the C2-iSH2 domain interface. Similar results were seen in soft agar and focus-formation assays, where p110β was similar to p110α-N345K in transforming potential. Inhibition of p110β by p85 was enhanced by a K342N mutation in p110β, which led to decreased activity in vitro, decreased basal Akt and ribosomal protein S6 kinase (S6K1) activation, and decreased transformation in NIH 3T3 cells. Moreover, unlike wild-type p110β, p110β-K342N was differentially regulated by wild-type and mutant p85, suggesting that the inhibitory C2-iSH2 interface is functional in this mutant. This study shows that the enhanced transforming potential of p110β is the result of its decreased inhibition by p85, due to the disruption of an inhibitory C2-iSH2 domain interface.

Class I PI3-kinases signal downstream of receptor tyrosine kinases and G protein-coupled receptors and have been implicated in tumorigenesis. Although the oncogenic potential of the PI3-kinase subunit p110α requires its mutational activation, other p110 isoforms can induce transformation when overexpressed in the wild-type state. In wild-type p110α, N345 in the C2 domain forms hydrogen bonds with D560 and N564 in the inter-SH2 (iSH2) domain of p85, and mutations of p110α or p85 that disrupt this interface lead to increased basal activity and transformation. Sequence analysis reveals that N345 in p110α aligns with K342 in p110β. This difference makes wild-type p110β analogous to a previously described oncogenic mutant, p110α-N345K. We now show that p110β is inhibited by p85 to a lesser extent than p110α and is not differentially inhibited by wild-type p85 versus p85 mutants that disrupt the C2-iSH2 domain interface. Similar results were seen in soft agar and focus-formation assays, where p110β was similar to p110α-N345K in transforming potential. Inhibition of p110β by p85 was enhanced by a K342N mutation in p110β, which led to decreased activity in vitro, decreased basal Akt and ribosomal protein S6 kinase (S6K1) activation, and decreased transformation in NIH 3T3 cells. Moreover, unlike wild-type p110β, p110β-K342N was differentially regulated by wild-type and mutant p85, suggesting that the inhibitory C2-iSH2 interface is functional in this mutant. This study shows that the enhanced transforming potential of p110β is the result of its decreased inhibition by p85, due to the disruption of an inhibitory C2-iSH2 domain interface. [PUBLICATION ABSTRACT]

Class I PI3-kinases signal downstream of receptor tyrosine kinases and G protein-coupled receptors and have been implicated in tumorigenesis. Although the oncogenic potential of the PI3-kinase subunit p110α requires its mutational activation, other p110 isoforms can induce transformation when overexpressed in the wild-type state. In wild-type p110α, N345 in the C2 domain forms hydrogen bonds with D560 and N564 in the inter-SH2 (iSH2) domain of p85, and mutations of p110α or p85 that disrupt this interface lead to increased basal activity and transformation. Sequence analysis reveals that N345 in p110α aligns with K342 in p110β. This difference makes wild-type p110β analogous to a previously described oncogenic mutant, p110α-N345K. We now show that p110β is inhibited by p85 to a lesser extent than p110α and is not differentially inhibited by wild-type p85 versus p85 mutants that disrupt the C2–iSH2 domain interface. Similar results were seen in soft agar and focus-formation assays, where p110β was similar to p110α-N345K in transforming potential. Inhibition of p110β by p85 was enhanced by a K342N mutation in p110β, which led to decreased activity in vitro, decreased basal Akt and ribosomal protein S6 kinase (S6K1) activation, and decreased transformation in NIH 3T3 cells. Moreover, unlike wild-type p110β, p110β-K342N was differentially regulated by wild-type and mutant p85, suggesting that the inhibitory C2–iSH2 interface is functional in this mutant. This study shows that the enhanced transforming potential of p110β is the result of its decreased inhibition by p85, due to the disruption of an inhibitory C2–iSH2 domain interface.