scholarly journals Effects of protein kinase inhibitors and protein phosphatase inhibitors on cyclic AMP-dependent down-regulation of vesicular monoamine transport in pheochromocytoma PC12 cells

FEBS Letters ◽  
1995 ◽  
Vol 368 (3) ◽  
pp. 411-414 ◽  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Pc12 Cells ◽  
Protein Phosphatase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Phosphatase Inhibitors ◽  
Monoamine Transport ◽  
Protein Phosphatase Inhibitors ◽  
Pheochromocytoma Pc12

scholarly journals Effects of protein phosphatase and kinase inhibitors on the cardiac L-type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase.

1995 ◽  
Vol 106 (3) ◽  
pp. 393-414 ◽  
Author(s):  
H C Hartzell ◽  
Y Hirayama ◽  
J Petit-Jacques
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Phosphatase ◽  
Kinase Inhibitors ◽  
Delayed Rectifier ◽  
Internal Perfusion ◽  
Ca Current ◽  
Phosphatase Inhibitors ◽  
Pkc Inhibitors ◽  
Kinase A

We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10-30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.

Phosphorylation regulates the assembly of NuMA in a mammalian mitotic extract

1997 ◽  
Vol 110 (11) ◽  
pp. 1287-1297 ◽  
Author(s):  
A. Saredi ◽  
L. Howard ◽  
D.A. Compton
Keyword(s):  
Mitotic Spindle ◽  
Kinase Inhibitors ◽  
Cultured Cells ◽  
Protein Kinase Inhibitors ◽  
Structural Function ◽  
Interphase Nuclei ◽  
Phosphatase Inhibitors ◽  
Insoluble Particles ◽  
Protein Phosphatase Inhibitors ◽  
Protein Dephosphorylation

NuMA is a 236 kDa nuclear protein that is required for the organization of the mitotic spindle. To determine how NuMA redistributes in the cell during mitosis, we have examined the behavior of NuMA in a mammalian mitotic extract under conditions conducive to the reassembly of interphase nuclei. NuMA is a soluble protein in mitotic extracts prepared from synchronized cultured cells, but forms insoluble structures when the extract becomes non-mitotic (as judged by the inactivation of cdc2/cyclin B kinase and the disappearance of mpm-2-reactive antigens). These NuMA-containing structures are irregularly shaped particles of 1–2 microm in diameter and their assembly is specific because other nuclear components such as the lamins remain soluble in the extract under these conditions. NuMA is dephosphorylated during this assembly process, and the assembly of these NuMA-containing structures is catalyzed by protein dephosphorylation because protein kinase inhibitors enhance their formation and protein phosphatase inhibitors block their formation. Finally, immunodepletion demonstrates that NuMA is an essential structural component of these insoluble particles, and electron microscopy shows that the particles are composed of a complex interconnected network of foci. These results demonstrate that phosphorylation regulates the solubility of NuMA in a mammalian mitotic extract, and the spontaneous assembly of NuMA into extensive structures upon dephosphorylation supports the conclusion that NuMA serves a structural function.

Effect of H-7, A Protein Kinase Inhibitor, on Cyclic AMP-Dependent Inhibition of Monoamine Transport in PC12 Cells

Pteridines ◽  
1995 ◽  
Vol 6 (3) ◽  
pp. 126-128
Author(s):  
Nobuo Nakanishi ◽  
Reiko Matsumoto ◽  
Kinji Kurihara ◽  
Takao Ueha ◽  
Hiroyuki Hasegawa
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Pc12 Cells ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitor ◽  
Monoamine Transport ◽  
Dependent Inhibition

scholarly journals Signalling pathways involved in the control of sperm cell volume

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 61-73 ◽  
Author(s):  
A M Petrunkina ◽  
R A P Harrison ◽  
M Tsolova ◽  
E Jebe ◽  
E Töpfer-Petersen
Keyword(s):  
Ion Channels ◽  
Cell Volume ◽  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Regulatory Volume Decrease ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Negative Effect ◽  
Volume Measurements

The ability to maintain cellular volume is an important general physiological function, which is achieved by specific molecular mechanisms. Hypotonically induced swelling results in the opening of K+and Cl−ion channels, through which these ions exit with accompanying water loss. This process is known as regulatory volume decrease (RVD). The molecular mechanisms that control the opening of the ion channels in spermatozoa are as yet poorly understood. The present study investigated pathways of osmo-signalling using boar spermatozoa as a model. Spermatozoa were diluted into isotonic and hypotonic Hepes-buffered saline in the presence or absence of effector drugs, and at predetermined intervals volume measurements were performed electronically. Treatment with protein kinase C (PKC) inhibitors staurosporine, bismaleimide I and bismaleimide X led to dose-dependent increases of both isotonic and hypotonic volumes (P<0.05). However, as the isotonic volume was affected more than the hypotonic volume, the kinase inhibitors appeared to improve RVD, whereas activation of PKC with phorbol dibutyrate blocked RVD. The increase in isotonic cell volume induced by bismaleimide X was observed in chloride-containing medium but not in the medium in which chloride was replaced by sulphate, implying that PKC was involved in the control of chloride channel activity, e.g. by closing the channel after volume adjustment. The protein phosphatase PP1/PP2 inhibitors calyculin and okadaic acid increased the isotonic volume only slightly but they greatly increased the relative cell volume and blocked RVD. The activation of RVD processes was found to be cAMP-dependent; incubation with forskolin and papaverine improved volume regulation. Moreover, papaverine was able to overcome the negative effect of protein phosphatase inhibitors. The mechanism of sperm RVD appears to involve (a) alterations in protein phosphorylation/dephosphorylation balance brought about by PKC and PP1 and (b) a cAMP-dependent activating pathway.

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