Differential Effects of Phosphatase Inhibitors on the Calcium Homeostasis and Migration of HaCaT Keratinocytes

Ruzsnavszky, Olga; Dienes, B.; Oláh, Tamás; Vincze, János; Gáll, Tamás; Balogh, Enikő; Nagy, Gábor; Bátori, Róbert; Lontay, Beáta; Erdõdi, Ferenc; Csernoch, László

doi:10.1371/journal.pone.0061507

Cited by 5 publications

(1 citation statement)

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“…One possibility is that the suppression is a result of T305/T306 phosphorylation that inhibits CaM binding [ 9 ]. It is also possible that Calyculin A leads to a suppression of the Ca 2+ rise during uncaging by regulating NMDA channels [ 37 ] or processes involved in Ca 2+ homeostasis [ 38 ]. In any case, it seems clear that the suppression of the Camui response to uncaging is not a result of occlusion by the elevation of the basal lifetime because, as mentioned above, it was not observed in the T286D/T305A/T306A mutant or in experiments with expressible PP1 and PP2A inhibitors, which produced comparable increase in the basal lifetime but no change in the magnitude of response to uncaging.…”

Section: Discussionmentioning

confidence: 99%

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

2015

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Because CaMKII is the critical Ca2+ sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca2+ falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.

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Section: Discussionmentioning

confidence: 99%