Regulation of Gβγi-Dependent PLC-β3 Activity in Smooth Muscle: Inhibitory Phosphorylation of PLC-β3 by PKA and PKG and Stimulatory Phosphorylation of Gαi-GTPase-Activating Protein RGS2 by PKG

In gastrointestinal smooth muscle, agonists that bind to G_i-coupled receptors activate preferentially PLC-β3 via Gβγ to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP₃) leading to IP₃-dependent Ca²⁺ release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-β3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A₁ receptor agonist, caused an increase in PI hydrolysis in a concentration-dependent fashion; stimulation was blocked by expression of the carboxyl-terminal sequence of GRK2(495–689), a Gβγ-scavenging peptide, or Gα_i minigene but not Gα_q minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-β3 and inhibited CPA-induced PI hydrolysis, Ca²⁺ release, and muscle contraction. The effect of isoproterenol on all three responses was inhibited by PKA inhibitor, myristoylated PKI, or AKAP inhibitor, Ht-31, whereas the effect of GSNO was selectively inhibited by PKG inhibitor, Rp-cGMPS. GSNO, but not isoproterenol, also phosphorylated Gα_i-GTPase-activating protein, RGS2, and enhanced association of Gα_i3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gα_i mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gβγ_i-dependent PLC-β3 activity by direct phosphorylation of PLC-β3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gα_i-GTP. This allows RGS2 to accelerate Gα_i-GTPase activity, enhance Gαβγ_i trimer formation, and inhibit Gβγ_i-dependent PLC-β3 activity.     

Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2β1

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.     

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (T_reg) cells by enhancing expression of Foxp3 in CD4⁺ T cells and suppressed effector helper T (T_H) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.     

Phosphorylation of LSD1 by PKCα Is Crucial for Circadian Rhythmicity and Phase Resetting

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Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.     

Phosphorylation and Inactivation of Glycogen Synthase Kinase 3β (GSK3β) by Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A)

Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr³⁵⁶ residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)³⁵⁶-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β.     

Direct Binding and Regulation of RhoA Protein by Cyclic GMP-dependent Protein Kinase Iα

Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1–44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1–59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.     

Protein Kinase Cδ-Mediated Phosphorylation of Phospholipase D Controls Integrin-Mediated Cell Spreading

Integrin signaling plays critical roles in cell adhesion, spreading, and migration, and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to the proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin are dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here we demonstrate that protein kinase Cδ (PKCδ) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCδ is activated and translocated to the edges of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCδ activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCδ and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation, and cell spreading after integrin activation. Together, these results suggest that PKCδ is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.Integrin-mediated cell adhesion, spreading, and migration, which are essential for cellular differentiation, proliferation, survival, chemotaxis, and wound healing, require cell polarization with an environmental stimulus (32). To regulate these integrin-mediated functions accurately, coordinated and spatial control of localized cytoskeletal rearrangement is required. The key downstream signaling molecules of integrin-mediated actin cytoskeletal rearrangements include small GTPases of the Rho family, such as Rho, Cdc42, and Rac (57, 58). Recently it was suggested that integrin indirectly regulates the recruitment of small G proteins and their localized activation at a specific plasma membrane region called the cholesterol-enriched membrane microdomain. Furthermore, the membrane targeting of these molecules appears to be required for the activation of downstream effectors that induce actin reorganization (8, 9, 48). However, in the absence of integrin signaling, despite the GTP loading status, activated Rac and Cdc42 remain in the cytosol and cannot activate downstream effectors, such as p21-activated kinase (PAK) (8). This regulation of the localization of small GTPases to a specific site is supported by the observation that Rac1 is localized and activated at the leading edges of migrating cells, while Cdc42 is also activated in cellular protrusions and in the peripheral region (33, 51). The differentially localized activation of small GTPases results in coordinated spatially confined signaling leading to cytoskeletal rearrangement, which is critical for the regulation of integrin-mediated cell spreading and directional cell migration.The hydrolysis of phosphatidylcholine by phospholipase D1 (PLD1) and PLD2 generates the messenger lipid phosphatidic acid (PA) in response to a variety of signals, which include hormones, neurotransmitters, and growth factors (17). It has been shown that PA affects actin cytoskeletal rearrangement and hence lamellipodium extension and integrin-mediated cell spreading as well as migration. PLD activity has been found in detergent-insoluble membrane fractions in which a wide variety of cytoskeletal proteins, such as F-actin, α-actinin, vinculin, paxillin, and talin, were enriched (34). Furthermore, the stimulation of PLD with physiologic and pharmacologic agonists results in its association with actin filaments (34). In addition, actin polymerization and stress fiber formation are tightly coupled to the activation of PLD (14). The formation of lamellipodium structures and membrane ruffles is blocked by PLD inhibition (53, 60), and PLD activity is critical for epithelial cell, leukocyte, and neutrophil adhesion and migration (41, 43, 52). Furthermore, we have previously shown that the activity of PLD is upregulated, and that the activated PLD is translocated to lamellipodia, after integrin activation (3). The PLD product PA acts as a lipid anchor for the membrane translocation of Rac, and this PA-mediated localized activation of Rac is critical for integrin-mediated cell spreading and migration through Rac downstream signaling activation and actin cytoskeleton rearrangement (3). However, the mechanisms that regulate the activation and localization of PLD, which induce the localized downstream activation of integrin signaling, have not been elucidated.Members of the protein kinase C (PKC) family of serine-threonine kinases are known to play important roles in the transduction of signals from the activation of integrin to cell adhesion and spreading, as well as in cell migration via actin reorganization (11, 25, 61, 66). Several studies have shown that the activities of several PKC isozymes are modulated and are crucially required for integrin-mediated cell spreading and migration. The PKCα, -δ, and -ɛ isotypes were activated and then translocated from the cytosol to the membrane after integrin activation, and inhibition of these PKC isozymes prevented cell spreading (10, 47, 66). In addition, the activation of PKCα, -δ, and -ɛ rescued the spreading of α5 integrin-deficient cells on fibronectin (10), and PKCβΙ mediated platelet cell spreading and migration on fibrinogen (2). It has also been demonstrated that PKCθ activity is involved in endothelial cell migration (65). These results suggest that the kinase activities of diverse members of PKC are involved in the integrin-mediated signaling pathway leading to the actin cytoskeletal rearrangement required for cell spreading and migration. Several PKC substrates are known to influence the actin cytoskeleton directly (42). However, the natures of the isoform-specific functions of PKC members and of their specific downstream effectors for actin cytoskeletal rearrangement induction by integrin signaling remain to be elucidated.In this study, we found that PKCδ is an upstream modulator of localized PLD activation in the integrin signaling pathway. We demonstrate for the first time that PKCδ activity (not PKCα or PKCɛ activity) is critical for integrin-mediated PLD activation, and we found that PLD2 is phosphorylated at Thr566 by PKCδ in the integrin signaling pathway. Furthermore, we show that this phosphorylation is critical for integrin-mediated targeting of PLD to membrane ruffles, Rac translocation to the membrane, and lamellipodium formation during cell spreading. These findings strongly suggest a bridge between PKCδ and the signaling of actin cytoskeletal rearrangement by the integrin signaling pathway via PLD activation, and they provide a novel molecular mechanism for localized PLD activation via PKCδ phosphorylation, which is critical for the actin cytoskeletal rearrangements required for integrin-mediated cell spreading.     

Spectroscopic Studies of GSK3β Phosphorylation of the Neuronal Tau Protein and Its Interaction with the N-terminal Domain of Apolipoprotein E

Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.     

Phosphorylation of LCRMP-1 by GSK3β promotes filopoda formation, migration and invasion abilities in lung cancer cells

LCRMP-1, a novel isoform of CRMP-1, can promote cancer cell migration, invasion and associate with poor clinical outcome in patients with non-small-cell lung cancer (NSCLC). However, the underlying regulatory mechanisms of LCRMP-1 in cancer cell invasiveness still remain obscure. Here, we report that GSK3β can phosphorylate LCRMP-1 at Thr-628 in consensus sequences and this phosphorylation is crucial for function of LCRMP-1 to promote filopodia formation, migration and invasion in cancer cells. Impediment of Thr-628 phosphorylation attenuates the stimulatory effects of LCRMP-1 on filopodia forming, migration and invasion abilities in cancer cells; simultaneously, kinase-dead GSK3β diminishes regulation of LCRMP-1 on cancer cell invasion. Furthermore, we also found that patients with low-level Ser-9-phosphorylated GSK3β expression and high-level LCRMP-1 expression have worse overall survival than those with high-level inactive GSK3β expressions and low-level LCRMP-1 expressions (P<0.0001). Collectively, these results demonstrate that GSK3β-dependent phosphorylation of LCRMP-1 provides an important mechanism for regulation of LCRMP-1 on cancer cell invasiveness and clinical outcome.     

Regulation of RhoA Signaling by the cAMP-dependent Phosphorylation of RhoGDIα

RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying G_s/cAMP-induced cross-talk with G_q/G₁₃/RhoA signaling.     

β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar¹,Ile⁴,Ile⁸]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.