scholarly journals Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability

2018 ◽  
Vol 314 (2) ◽  
pp. C233-C241 ◽  
Author(s):  
John D. Tompkins ◽  
Todd A. Clason ◽  
Thomas R. Buttolph ◽  
Beatrice M. Girard ◽  
Anne K. Linden ◽  
...  
Keyword(s):  
G Protein ◽  
Neuronal Excitability ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Src Family Kinases ◽  
Receptor Internalization ◽  
Src Family Kinase ◽  
Pac1 Receptor ◽  
Neuron Excitability ◽  
Endosomal Signaling

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

scholarly journals Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability

2017 ◽  
Vol 313 (2) ◽  
pp. C219-C227 ◽  
Author(s):  
Jean C. Hardwick ◽  
Todd A. Clason ◽  
John D. Tompkins ◽  
Beatrice M. Girard ◽  
Caitlin N. Baran ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Neuronal Excitability ◽  
Receptor Activation ◽  
Erk Signaling ◽  
Pac1 Receptor ◽  
Signaling Mechanism ◽  
Erk Activation ◽  
Neuron Excitability ◽  
Endosomal Signaling ◽  
Gated Channel

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5–10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels ( Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

Requirements of src family kinase activity associated with CD45 for myeloma cell proliferation by interleukin-6

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2172-2178 ◽  
Author(s):  
Hideaki Ishikawa ◽  
Naohiro Tsuyama ◽  
Saeid Abroun ◽  
Shangqin Liu ◽  
Fu-Jun Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Kinase Inhibitor ◽  
Myeloma Cell ◽  
Src Family Kinases ◽  
Src Family Kinase ◽  
Receptor Complexes ◽  
Intracellular Signals ◽  
Cellular Context

Abstract Specific intracellular signals mediated by interleukin-6 (IL-6) receptor complexes, such as signal transducer and activator of transcription 3 (STAT 3) and extracellular signal–regulated kinase (ERK) 1/2, are considered to be responsible for inducing a variety of cellular responses. In multiple myeloma, IL-6 only enhanced the proliferation of CD45+ tumor cells that harbored the IL-6–independent activation of src family kinases even though STAT3 and ERK1/2 could be activated in response to IL-6 in both CD45+ and CD45− cells. Furthermore, the IL-6–induced proliferation of CD45+ U266 myeloma cells was significantly suppressed by Lyn-specific antisense oligodeoxynucleotides or a selective src kinase inhibitor. These results indicate that the activation of both STAT3 and ERK1/2 is not enough for IL-6–induced proliferation of myeloma cell lines that require src family kinase activation independent of IL-6 stimulation. Thus, the activation of the src family kinases associated with CD45 expression is a prerequisite for the proliferation of myeloma cell lines by IL-6. We propose a mechanism for IL-6–induced cell proliferation that is strictly dependent upon the cellular context in myelomas.

Src Kinase Inhibitors Enhance ATRA-Mediated Differentiation of Myeloid Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4264-4264
Author(s):  
Michelle B. Miranda ◽  
Daniel E. Johnson
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Src Family Kinases ◽  
Myeloid Differentiation ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Current Standard ◽  
The Impact

Abstract Myeloid leukemias are characterized by a blockade in differentiation resulting in accumulation of proliferating progenitor cells and a lack of mature granulocytes and monocytes. In acute promyelocytic leukemia (APL), a subtype of AML, treatment with all-trans retinoic acid (ATRA) has been shown to overcome this blockade in differentiation and is the current standard of care for APL patients. However, other forms of myeloid leukemia show no response to ATRA. Src family kinases (SFK) have been shown to be activated during normal myeloid development by both proliferation-inducing cytokines, such as IL-3, and differentiation-promoting factors, such as G-CSF. To elucidate the role of src family kinases during myeloid differentiation, we examined the impact of SFK inhibitors, PP1 and PP2 on ATRA-mediated differentiation of myeloid cell lines. Interestingly, PP1 potentiated ATRA-mediated myeloid differentiation of HL-60 cells, inducing a two-fold increase in differentiated cells at 72 hours as determined by CD11b staining, NBT reduction and morphological analysis. To determine whether enhancement of ATRA-mediated differentiation was specific for PP1, or could be promoted by a different src family kinase inhibitor, we evaluated the impact of PP2. PP2 was found to exhibit similar effects on ATRA-induced myeloid differentiation of HL-60 cells. Additionally, both PP1 and PP2 enhanced ATRA-induced monocytic differentiation of U937 cells and granulocytic differentiation of NB-4 cells. Interestingly, PP1 was found to enhance ATRA-induced differentiation even when added 24 hours after addition of ATRA. The impact of PP1 on ATRA-mediated myeloid differentiation was dependent on signaling via the MEK/ERK pathway since pre-treatment with U0126, a MEK-1/-2 inhibitor, attenuated myeloid differentiation induced by the combination of RA and PP1. Reporter assays utilizing an RARE-luciferase construct indicate that PP1 and PP2 do not enhance ATRA-mediated differentiation by promoting the transcriptional activity of the retinoic acid receptor (RAR). Together, our results demonstrate that PP1 and PP2 potently enhance ATRA-induced myeloid differentiation of AML cell lines and suggest that src inhibition in combination with ATRA may be a useful treatment for AML. Studies evaluating the impact of src inhibition on ATRA-mediated differentiation of primary AML cells are currently underway.

scholarly journals Src-family kinases mediate an outside-in signal necessary for β2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

2006 ◽  
Vol 396 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Licia Totani ◽  
Antonio Piccoli ◽  
Stefano Manarini ◽  
Lorenzo Federico ◽  
Romina Pecce ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Chinese Hamster Ovary Cells ◽  
Src Family Kinases ◽  
Cell Suspensions ◽  
Polymorphonuclear Cells ◽  
Polymorphonuclear Leucocytes ◽  
Dependent Manner ◽  
Src Family Kinase ◽  
Β2 Integrin ◽  
Β2 Integrins

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin–IgG fusion protein, by a mechanism that involved β2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(β2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an ‘outside-in’ signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented β2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin–IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck−/−fgr−/− and hck−/−fgr−/−lyn−/− mice showed significant impairment of β2-integrin-mediated adhesion to CHO-E. Moreover, the expression of β2 integrin activation epitopes at the sites of cell–cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a β2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for β2 integrin function in PMN tethered by E-selectin.

scholarly journals Activation of MEK/ERK signaling contributes to the PACAP-induced increase in guinea pig cardiac neuron excitability

2016 ◽  
Vol 311 (4) ◽  
pp. C643-C651 ◽  
Author(s):  
John D. Tompkins ◽  
Todd A. Clason ◽  
Jean C. Hardwick ◽  
Beatrice M. Girard ◽  
Laura A. Merriam ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Neuronal Excitability ◽  
Low Voltage ◽  
Ionic Currents ◽  
Erk Signaling ◽  
Repetitive Firing ◽  
Neuronal System ◽  
Cellular Mechanisms ◽  
Pac1 Receptor ◽  
Neuron Excitability

Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides ( Adcyap1) signaling at the selective PAC1 receptor ( Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels ( Ih) and another flowing through low-voltage-activated calcium channels ( IT), which support the peptide-induced increase in excitability. Thus a PACAP - and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.

scholarly journals Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3410-3414 ◽  
Author(s):  
Analia Garcia ◽  
Todd M. Quinton ◽  
Robert T. Dorsam ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Kinase Inhibitor ◽  
Thromboxane A2 ◽  
Receptor Activation ◽  
Src Family Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Src Family Kinase

AbstractThe binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non–aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.

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