Protein phosphatase type-2C isozymes present in vertebrate retinae: Purification, characterization, and localization in photoreceptors

Klumpp, Susanne; Selke, Dagmar; Fischer, Dietmar; Baumann, Arnd; Müller, Frank; Thanos, Solon

doi:10.1002/(sici)1097-4547(19980201)51:3<328::aid-jnr6>3.0.co;2-i

Cited by 24 publications

(15 citation statements)

References 31 publications

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“…Inhibition of the [ 32 P]G-CAP-2 dephosphorylation occurred only when Mg 2ϩ was removed from the reaction mixture by EDTA. EDTA is the only inhibitor of the fourth type of protein phosphatase, the Mg 2ϩ -dependent phosphatase PP2C, which is also present in the retina (30) (Fig. 2, A, C, and D).…”

Section: C-terminal Ser 201 In Gcap-2 Undergoes Phosphorylation By Cndpkmentioning

confidence: 97%

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Peshenko

Dizhoor

2004

Journal of Biological Chemistry

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Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca 2؉ but activate retGC when Ca 2؉ dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca 2؉ . We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotidedependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser 201 , had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca 2؉ strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca 2؉ -insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca 2؉ -sensitive manner. The Ca 2؉ -dependent conformational changes in GCAP-2 affect the areas around Glu 62 residue in the entering helix of EFhand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu 136 -Glu 138 between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser 201 from the constraint caused by the Ca 2؉ -bound conformation.Photoisomerized rhodopsin triggers hydrolysis of cGMP through the activation of the G-protein transducin, which stimulates cGMP phosphodiesterase and thus causes cGMPgated channels to close (see Refs. 1-3 for review). Rods and cones rapidly recover to their resting potential after excitation induced by a brief non-saturating exposure to light. They also adapt to a constant background illumination by decreasing the amplitude and accelerating the kinetics of their electrical responses to a standard test flash (see Refs. 1-3 for review). Reopening of the cGMP-gated channels during recovery and light adaptation requires acceleration of cGMP synthesis by guanylyl cyclase, a process controlled by the level of intracellular free Ca 2ϩ . In the dark, Ca 2ϩ extruded from the photoreceptor outer segment by a Na ϩ /K ϩ ,Ca 2ϩ exchanger re-enters the outer segment through the open cGMP-gated channels so that the free intracellular outer segment Ca 2ϩ concentrations reach 250 nM in mammals and 350 -450 nM in lower vertebrates (4 -6). In the light, cGMP is hydrolyzed, and these channels close while the exchanger continues to extrude Ca 2ϩ ions from the photoreceptor outer segment; therefore, Ca 2ϩ concentrations decrease nearly 10-fold (4 -7). Ca 2ϩ -binding proteins GCAP-1 1 and GCAP-2 activate retGC1 and retGC2 (and their homologs found in photoreceptors of different vertebr...

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Section: C-terminal Ser 201 In Gcap-2 Undergoes Phosphorylation By Cndpkmentioning

confidence: 97%

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Peshenko

Dizhoor

2004

Journal of Biological Chemistry

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“…For example, they act as negative regulators of the pheromone-inducible MAPK pathway in yeast, and PP2C enzymes are also involved in cell division and synchronizing budding and mitochondrial multiplication in yeast (Meskiene et al 1998;Roeder et al 1998). In vertebrates the importance of PP2C has been demonstrated in the development of the retina, in the regulation of transmembrane conductance, and in liver glycogen metabolism (Ortmeyer 1997;Travis et al 1997;Klumpp et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum

et al. 1999

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Ten transcripts (Mpc1-10) homologous to protein phosphatases of the 2C family have been isolated from the halophyte Mesembryanthemum crystallinum (common ice plant). Transcripts range in size from 1.6 to 2.6 kb, and encode proteins whose catalytic domains are between 24% and 62% identical to that of the Arabidopsis PP2C, ABI1. Transcript expression is tissue speci®c. Two isoforms are present only in roots (Mpc1 and Mpc5), three in young leaves (Mpc6, 8 and 9), two in old leaves (Mpc6 and Mpc8), and two in post-¯ow-ering leaves (Mpc8 and Mpc9). Mpc2 is strongly expressed in roots and also in seeds, meristematic tissues and mature¯owers. Mpc3 is speci®c for leaf meristems, and Mpc4 is found in root and leaf meristems. Mpc7 is restricted to meristematic tissues. Mpc10 is only present in mature¯owers. Mpc2 (in roots and leaves), Mpc5 (in roots) and Mpc8 (weakly in leaves) are induced by salinity stress and drought conditions with dierent kinetics in dierent tissues, but other Mpcs are downregulated by stress. Cold stress (4°C) leads to a decline in Mpc5 and Mpc6, but low temperature provoked a longterm (days) increase in Mpc2 levels in leaves and a transient increase (less than 24 h) in roots. Four fulllength transcripts have been obtained. In each case, after over-expression in E. coli, the isolated proteins exhibited (Mg 2+ -dependent, okadeic acid-insensitive) protein phosphatase activity, although activity against 32 P-phosphocasein varied among dierent PP2Cs. Determination of tissue developmental and stress response speci®city of PP2C will facilitate functional studies of signal-transducing enzymes in this halophytic organism.

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“…The replacement of Mn 2+ in the catalytic center of tPphA with two divalent cations (Ni 2+ and Ca 2+ ) could also inhibit tPphA activity. The low IC50 value of Ni 2+ measured in this report suggests that this divalent cation is a stronger inhibitor of tPphA than Ca 2+ , which also shows inhibitory effects on other PP2Cs [30][31][32][33][34][35][36]. Other divalent cations have also been reported to inhibit PP2C catalytic activity [32,34,37].…”

Section: Inhibition Effects Of Nine Chemicals On Tppha Activitymentioning

confidence: 79%

Structural and Biochemical Characterization of a Cyanobacterial PP2C Phosphatase Reveals Insights into Catalytic Mechanism and Substrate Recognition

Yuan

Wang

et al. 2016

Catalysts

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PP2C-type phosphatases play roles in signal transduction pathways related to abiotic stress. The cyanobacterial PP2C-type phosphatase tPphA specifically dephosphorylates the PII protein, which is a key regulator in cyanobacteria adapting to nitrogen-deficient environments. Previous studies have shown that residue His39 of tPphA is critical for the enzyme's recognition of the PII protein; however, the manner in which this residue determines tPphA substrate specificity is unknown. Here, we solved the crystal structure of H39A, a tPphA variant. The structure revealed that the mutation of residue His39 to alanine changes the conformation and the flexibility of the loop in which residue His39 is located, and these changes affect the substrate specificity of tPphA. Moreover, previous studies have assumed that the FLAP subdomain and the third metal (M3) of tPphA could mutually influence each other to regulate PP2C catalytic activity and substrate specificity. However, despite the variable conformations adopted by the FLAP subdomain, the position of M3 was consistent in the tPphA structure. These results indicate that the FLAP subdomain does not influence M3 and vice versa. In addition, a small screen of tPphA inhibitors was performed. Sanguinarine and Ni 2+ were found to be the most effective inhibitors among the assayed chemicals. Finally, the dimeric form of tPphA was stabilized by cross-linkers and still exhibited catalytic activity towards p-nitrophenyl phosphate.

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Protein phosphatase type-2C isozymes present in vertebrate retinae: Purification, characterization, and localization in photoreceptors

Cited by 24 publications

References 31 publications

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum

Structural and Biochemical Characterization of a Cyanobacterial PP2C Phosphatase Reveals Insights into Catalytic Mechanism and Substrate Recognition

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