Mary D. Pato

Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR. Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by >90% but did not abolish it completely. PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability ( P o). Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision. PP2C and PP2A had distinct effects on channel gating; P o declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR. Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by > 90% but did not abolish it completely. PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability (Po). Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision. PP2C and PP2A had distinct effects on channel gating Po declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.

Myosin Light Chain Phosphatase and Kinase Abnormalities in Fetal Sheep Pulmonary Hypertension

Inasmuch as smooth muscle contractile protein abnormalities may account for the maintenance of a high pulmonary vascular resistance, we evaluated the pulmonary arterial myosin light chain kinase (MLCK) and phosphatase (MLCP) in normal and pulmonary hypertensive (PH) fetal sheep. In addition, aorta and vena cava MLCP and MLCK activities were also measured. The MLCK activity(nanomoles/min/mg) was determined by the incorporation of[32P]PO4-3 to the 20-kD smooth muscle myosin light chains and the MLCP activity by assaying for the dephosphorylation of the 20-kD myosin light chain (MLCP-light chain) and heavy meromyosin (MLCP-HMM). The MLCP content was determined by Western blot analysis. PH was characterized by a significant increase in the right-to-left ventricular wall weight ratio from 0.99 ± 0.04 in the control to 1.52 ± 0.12 in the experimental group (p < 0.01). The pulmonary MLCP-light chain and MLCP-HMM activities in the experimental group were 2.0 ± 0.2 and 1.3 ± 0.2 and significantly lower than in the control group values (3.8 ± 0.5 and 2.5 ± 0.3; p < 0.01). The MLCK activity was 9.6± 1.2 in the control and 7.8 ± 0.7 in the experimental fetal pulmonary artery (p = NS). The activities of both enzymes in the aorta and vena cava samples were not altered by PH. The MLCP content in experimental animals (0.50 ± 0.09 OD × mm2) was significantly lower than that for the control pulmonary tissue (1.72 ± 0.42; p < 0.01), suggesting that PH down-regulates pulmonary vascular MLCP expression. In conclusion, the maintenance of a high pulmonary vascular resistance in PH may be secondary to abnormalities in tissue content and/or activity of MLCP.

Comparison of the properties of the protein phosphatases from avian and mammalian smooth muscles: Purification and characterization of rabbit uterine smooth muscle phosphatases

Three protein phosphatases were purified to near homogeneity from rabbit uterine muscle. These enzymes are termed rabbit uterine smooth muscle phosphatase (RU SMP)-I, -II, and -IV. RU SMP-I is composed of three subunits (Mr 60,000, 55,000, and 38,000) which comigrated with the subunits of turkey gizzard smooth muscle phosphatase (TG SMP)-I. Ethanol treatment of RU SMP-I dissociated the subunits and led to the purification of its catalytic subunit (Mr 38,000), RU SMP-Ic. Structural homology between the turkey gizzard and rabbit uterine SMP-I is indicated by the cross-reactivity of RU SMP-I with the polyclonal antibodies against TG SMP-I and -Ic. Like TG SMP-II, RU SMP-II is inactive in the absence of divalent cations and can be activated by Mg2+ and Mn2+. However, their electrophoretic profiles on sodium dodecyl sulfate-polyacrylamide gel are different. RU SMP-II shows two bands (Mr 42,000 and 44,000) while TG SMP-II is monomeric (Mr 43,000). Western blot analysis revealed that the 42,000 and 44,000-Da proteins cross-react with anti-TG SMP-II antibodies, suggesting that these proteins share common structural properties. The anti-TG SMP-I and Ic antibodies do not cross-react with RU SMP-II and -IV. Likewise, the anti-TG SMP-II antibodies do not cross-react with RU SMP-I and -IV, implying that these enzymes are distinct. RU SMP-IV is composed of a catalytic subunit (Mr 40,000) and a subunit with a molecular weight of 60,000 or 58,000. All three rabbit uterine smooth muscle phosphatases dephosphorylate the isolated myosin light chains but only RU SMP-IV dephosphorylates heavy meromyosin. However, when the catalytic subunit of RU SMP-I is dissociated from the regulatory subunits, it is active toward heavy meromyosin and exhibits higher activity toward myosin light chains and phosphorylase a than its holoenzyme. The substrate specificity of these enzymes and the effects of ATP, NaF, pyrophosphate, okadaic acid, Mg2+, Mn2+, and Ca2+ on their activities are very similar to those of the turkey gizzard smooth muscle phosphatases.

Regulation of smooth muscle phosphatase-II by divalent cations

SummarySmooth Muscle Phosphatases II (SMP-I1) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg2+ and Mn2+ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg2+ is 1.5–2.5-fold higher than the maximal Mn2+ activation. However, the latter is achieved at a lower concentration than the maximal Mg2+-activation. Furthermore, at low cation concentrations (≲ 2 mM), the Mn2+-activated activity is higher than the Mg2+-activated activity. In the presence of both cations, the effect of Mn2+ predominates suggesting that the affinity of the enzyme for Mn2+ is greater than for Mg2+. In contrast to Mg2+ and Mn2+, Ca2+ does not activate SMP-II but it was observed to antagonize the effects of Mg2+ and Mn2+. Ca2+ acts as a competitive inhibitor of Mg2+. However, the inhibitory effect at high Ca2+ concentrations is not completely reversed by increasing the Mg2+ concentration. Mn2+ activation is also inhibited by Ca2+ but to a lesser extent. Ca2+ cannot completely inhibit Mn2+-activation suggesting that SMP-I1 has greater affinity for Mn2+ than for Ca2+. The finding that Ca2+ inhibits the activation of SMP-II raises the possibility that Ca2+ may be a regulator of SMP-II in vivo.

Protein phosphorylation in rat cardiac microsomes: Effects of inhibitors of protein kinase A and of phosphatases

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 µM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (Mr 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 µM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polyp eptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (Mr 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates. (Mol Cell Biochem 175: 109–115, 1997

[42] Purification of smooth muscle myosin phosphatase from Turkey gizzard

Publisher Summary This chapter describes the purification procedure of smooth muscle myosin phosphatase from turkey gizzard. The smooth muscle phosphatases are always prepared from fresh turkey gizzards. The phosphatase activity toward heavy meromyosin and the activity of SMP-I toward myosin light chains are reduced considerably. However, freezing of the 30-60% (NH 4 )SO 4 fraction of the extract from fresh turkey gizzards does not appear to change significantly the physical and enzymatic properties and activities of the phosphatases. Optimal resolution of smooth muscle phosphatase-I (SMP-I), SMP-II, and SMP-III on Sephacryl S-300 is observed when 30–35 ml of the sample is applied to the column (5 x100 cm). Application of a larger volume results in loss of resolution. Therefore, three successive runs are required to chromatograph the total sample (90 ml) obtained from the (NH 4 ) 2 SO 4 fractionation.

Purification and characterization of pregnant sheep myometrium myosin light chain kinase

Myosin light chain kinase (MLCK) has been purified from the myometrium of pregnant sheep. The Mr of the enzyme was determined from SDS-polyacrylamide gels to be 160,000. It requires Ca2+ and calmodulin for activation, and phosphorylates the 20,000-Da light chains of myosin at a rapid rate. The specific activity for the myosin light chains from turkey gizzards and rabbit uterine muscle are 7.7 and 5.4 mumol/min/mg, respectively. The Km for the former substrate is 40 microM and the Vmax of the reaction is 19 mumol/min/mg. Polyclonal antibodies raised against the enzyme cross-reacted with pregnant sheep myometrium (psm), turkey gizzard (tg), and chicken gizzard MLCK. Affinity purification of the antibodies on tg-MLCK Sepharose resulted in the preparation of two fractions of antibodies with different reactivity toward these proteins. Fraction A antibodies which did not bind to the affinity column cross-reacted only with psm-MLCK while Fraction B antibodies which bound to the column cross-reacted with all three proteins. Western blots of extracts of turkey gizzards, human myometrium, and various tissues from sheep showed cross-reactivity of both fractions of antibodies with a 160,000-Da protein in the extracts of sheep smooth muscles. Only Fraction B antibodies cross-reacted with a protein (130,000 Da) in turkey gizzards and human myometrium extracts. Prolonged tryptic digestion of psm-MLCK produced large fragments Mr approximately 60,000 which appears to be similar to that formed from tg-MLCK, and some smaller peptides. Fraction A antibodies cross-reacted with the small peptides while Fraction B antibodies cross-reacted with the large fragments but not vice versa. Further analysis of the tryptic peptides suggests that the epitopes of Fraction A antibodies are localized in a peptide which appears to be in the NH2-terminal region of the molecule.