PROTEIN PHOSPHATASES

SERINE/THREONINE PHOSPHATASES

CALCINEURIN (PP-2B)

Calcineurin (CaN) is the neuronal form of the widely distributed Ca²⁺/calmodulin-dependent Ser/Thr protein phosphatase 2B (PP-2B). CaN is a heterodimer consisting of a catalytic A subunit (57-61 kDa) and a regulatory B subunit (19 kDa). The catalytic A subunit is composed of four functional domains: the catalytic core with sequence homology to PP-1 and PP-2A (located between residues 71-235 in the rat brain ad isoform); binding sites for both calmodulin (residues 391-414) and CaN B-regulatory subunit; and a C-terminal (residues 457-482) autoinhibitory domain.

The role of the autoinhibitory domain was revealed by experiments employing limited proteolysis of calcineurin. The truncated catalytic A subunit was still capable of binding the regulatory B subunit and CaM, however it no longer required Ca²⁺ or CaM for full activity^1,2. Also, addition of a polypeptide with the deduced autoinhibitory sequence inhibited CaN phosphatase activity in a competitive manner (see catalog #PR-104, below)^3,4. Deletion mutations of CaN have shown that the autoinhibitory region is also involved in regulating binding of the FK506-FKBP12 complex to CaN^4,5.


CALCINEURIN ENZYME

SE-162
Calcineurin AaB (human, recombinant)
MW=60 and 19 kDa, Storage: -70°C
Recombinant human protein expressed in a baculovirus expression system. Highly active enyzme.
Inquire


SE-112
Calcineurin, PP2B (from bovine brain)
Calcium/calmodulin-activated protein phosphatase
MW=60 and 20 kDa Storage: -20°C
Supplied partially purified, lyophilized. Specific activity: 2.5-5 kU/mg protein. One unit will cause 50% inhibition of activated phosphodiesterase in the presence of calmodulin and 100 µM calcium at pH 7.5 and 30°C^1,2.
100 U
500 U


CALCINEURIN SUBSTRATES

P-118
[Ala-97]-RII (81-99), PKA substrate for radioactive assays
Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Ala-Ala-Glu
>97%, MW=2112.4, Storage: -20°C
Sequence is from cAMP-dependent protein kinase (PKA) regulatory subunit type II (RII). The P³²-labeled peptide has been widely used as a phosphoserine substrate for calcineurin in radioactive assays. K_m =25 µM, V_max=6 µmol/min/mg using bovine brain CaN^3,4,5.
0.5 mg
5 x 0.5 mg

P-160
RII Phosphopeptide, Calcineurin substrate for non-radioactive assays
Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-pSer-Val-Ala-Ala-Glu
>97%, MW=2192.0, Storage: -20°C
Sequence is the same as P-118 above but with a phosphoserine at Ser-95. This peptide can be used directly as a calcineurin (protein phosphatase 2B, PP2B) substrate^3,5,6. Phosphate release can be quantified using the malachite green colorimetric assay^7,8.
0.5 mg
5 x 0.5 mg


CALCINEURIN INHIBITORS

See Table 6 below for inhibitor specificity.
See "IMMUNOPHILINS" for additional potent calcineurin inhibitors

PR-104
Calcineurin Autoinhibitory Peptide (CN412)
Ile-Thr-Ser-Phe-Glu-Glu-Ala-Lys Gly-Leu-Asp-Arg-Ile-Asn-Glu-Arg-Met-Pro-Pro-Arg-Arg-Asp-Ala-Met Pro
>97%, MW=2930.7, Lyophilized. Storage: -20°C
Sequence is calcineurin (CaN) autoinhibitory domain (rat brain CaN Aa residues 457-482).
It inhibits calcineurin with an IC₅₀ of 10 µM using [³²P]myosin light chain as substrate⁹. Inhibits Mn²⁺-stimulated calcineurin activity but has no effect on Ni²⁺-stimulated activity¹⁰. K_i=4.4 µM with radiolabeled [Ala-97]-RII (81-99) peptide (P-118) as the phosphatase substrate⁵.
0.5 mg
5 x 0.5 mg
NEW LOWER PRICE

PR-100
Cypermethrin
96%, MW=416.3 [52315-07-8] Storage: RT
Synthetic type II pyrethroid. Potent calcineurin (PP2B) inhibitor IC₅₀ ~0.03 nM¹¹. May also have varied effects on ion channels¹². A unique new tool for studying the role of calcineurin in signal transduction.
10 mg
50 mg

PR-101
Deltamethrin
99%, MW=505.2 [52918-63-5] Storage: RT
Synthetic type II pyrethroid. Potent calcineurin (PP2B) inhibitor IC₅₀ ~0.03 nM¹¹. May also have varied effects on ion channels¹².
10 mg
50 mg

PR-103
Permethrin
97%, MW=391.3 [52645-53-1] Storage: RT
Synthetic type I pyrethroid. Structurally similar to the calcineurin inhibitors cypermethrin and deltamethrin but weakly active or inactive depending on assay conditions¹¹. May be used as a negative control for cypermethrin and deltamethrin.
10 mg
50 mg

PR-102
Fenvalerate
99%, MW=419.9 [51630-58-1] Storage: RT
Synthetic type II pyrethroid. Potent calcineurin (PP2B) inhibitor IC₅₀ ~20 nM¹¹. May also have varied effects on ion channels¹².
20 mg
100 mg


CALCINEURIN ANTIBODY

SA-212
Anti-Calcineurin B
Rabbit polyclonal antibody. Immunogen: Full length recombinant mouse calcineurin B (CnB) expressed in E. coli. Supplied as rabbit serum containing sodium azide. Recognizes CnB 19 kDa regulatory subunit protein in human, rat and mouse. Applications: WB 1/2000, IC 1/500 and IH 1/500.
100 µl


SERINE/THREONINE PHOSPHATASE ENZYMES

SE-119
Protein phosphatase 2A₁, PP2A₁ (from bovine kidney)
>95%, MW=36, 55, 60 kDa (EC 3.1.3.16) Storage: -20°C
Supplied in 10% glycerol buffer. Specific activity: 750 U/mg enzyme. One unit will hydrolyze 1 nmol phosphate from phosphorylase per minute at pH 7.0 and 30°C.
1 µg

SE-120
Protein phosphatase 2A₂, PP2A₂ (from bovine kidney)
>95%, MW=36 and 60 kDa (EC 3.1.3.16) Storage: -20°C
Supplied in 10% glycerol buffer. Specific activity: 750 U/mg protein. One unit will hydrolyze 1 nmol phosphate from phosphorylase per minute at pH 7 and 30°C^13,14.
1 µg


SERINE/THREONINE PHOSPHATASE INHIBITORS

TABLE 6. SELECTIVITY OF PROTEIN PHOSPHATASE INHIBITORS

EI-192
Calyculin A
98%, MW=1009.2 [101932-71-2] Storage: -20°C
Calyculin A is a structurally unique marine toxin with potent and specific protein phosphatase inhibiting activity (see references and for reviews on protein phosphatases). It inhibits PP-2A with similar potency to okadaic acid (IC₅₀=0.5-1.0 nM) but inhibits PP-1 with a 10- to 100-fold greater potency¹⁸. Calyculin A stimulates the contraction of smooth muscle¹⁹, induces intracellular protein phosphorylation in cultured human keratinocytes²⁰ and is a potent tumor promotor²¹. Inhibits apoptosis²².
25 µg
100 µg

PR-106
Cantharidic Acid
98%, MW=214.2 [28874-45-5] Storage: RT
Potent and selective inhibitor of PP2A, IC₅₀=53 nM (IC₅₀ for PP1=562 nM^23,24. Useful for inhibiting PP1 and PP2A during purification of phosphorylated proteins.
10 mg
50 mg

PR-105
Cantharidin
98%, MW=196.2 [56-25-7] Storage: RT
Potent and selective inhibitor of PP2A, IC₅₀=40 nM (IC₅₀ for PP1=473 nM)^23,24. May be hydrolyzed to cantharidic acid in aqueous medium. Useful for inhibiting PP1 and PP2A during purification of phosphorylated proteins.
20 mg
100 mg

PR-108
1,4-Dimethylendothall
98%, MW=214.2 [109282-27-1] Storage: RT
Close structural analog of cantharidins and endothall but inactive or weakly active (does not inhibit PP2A at 250 nM). May be used as a negative control compound for endothall, cantharidin and cantharidic acid²⁴.
10 mg
50 mg

PR-107
Endothall
98%, MW=186.2 [145-73-3] Storage: RT
Inhibits PP2A with intermediate potency IC₅₀=970 nM (IC₅₀ for PP1=5000 nM)^23,24.
20 mg
100 mg

EI-193
Microcystin LR
98%, MW=994.0 [101043-37-2] Storage: -20°C
Microcystin LR is a hepatotoxic, cyclic peptide obtained from cyanobacteria²⁵. It contains a unique C₂₀ amino acid, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid. It is a potent inhibitor of PP-1 (IC₅₀=1.7 nM) and even more potent for PP-2A (IC₅₀=0.04 nM)²⁶. It does not induce any effects on mouse skin or human fibroblasts¹⁶ due to cell membrane impermeability. It is however absorbed by hepatocytes²⁷ via the multispecific organic anion transporter²⁸.
500 µg
5 x 500 µg

EI-194
Nodularin
95%, MW=824.9 [118399-22-7] Storage: -20°C
Nodularin is a hepatotoxic, cyclic peptide related to microcystin LR²⁵ with a similar activity profile²⁹. It does not induce any effects on mouse skin or human fibroblasts³⁰.
500 µg
5 x 500 µg

EI-181
Okadaic acid
98%, MW=805.0 [78111-17-8] Storage: -20°C
Okadaic acid is a naturally occurring polyether toxin produced by marine dinoflagellates. It is a potent and selective inhibitor of protein phosphatases inhibiting PP-2A completely at 1 nM and PP1 at higher concentrations (IC₅₀=10-15 nM). PP-2B is much less sensitive to okadaic acid than PP1, while PP-2C is not inhibited. This selectivity is the basis for an improved identification and quantification procedure for these enzymes³¹. The hydrophobic backbone of okadaic acid enables it to enter cells where it stimulates intracellular protein phosphorylation³². It mimics the effects of insulin³³, enhances transmitter release at neuromuscular junctions³⁴, causes vasodilation³⁵ and is a very potent tumor promotor³⁶. Okadaic acid is an extremely useful tool for studying cellular processes that are regulated by phosphorylation³⁷.
25 µg
100 µg

EI-250
1-nor-Okadaone
98%, MW=759.0 [131204-29-0] Storage: -20°C
Negative control for okadaic acid³⁸.
25 µg

SE-130
Protein Phosphatase Inhibitor 2 (rabbit, recombinant)
MW=22.8 kDa, >95% by SDS-PAGE, Storage: -70°C
Heat-stable protein from rabbit skeletal muscle expressed in E. coli³⁹. It is a specific inhibitor of protein phosphatase 1 (PP1). Specific activity is ~5000 U/mg where one unit will inhibit 1 U of PP1 (1 nmol phosphate released/min at 30°C from phosphorylase a) by 50%⁴⁰. 1 µg can inhibit >90% PP1 phosphatase activity in cell extracts⁴¹. Supplied at 1 mg/ml.
10 µg
5 x 10 µg

EI-243
Tautomycin
97%, MW=766.9 [109946-35-2] Storage: -20°C
Tautomycin is a potent and selective protein phosphatase inhibitor isolated from Streptomyces verticillatus⁴². It inhibits PP1 completely at 3 nM and PP2A at 30 nM⁴³. Inhibition of PP2B is over 10,000-fold weaker and PP2C is not inhibited. Tautomycin shares some structural similarity with okadaic acid but has opposite relative potency for PP1 and PP2A. Its cell permeability and unique inhibitory profile make tautomycin a useful tool for identifying phosphatase subtypes and physiological substrates for PP1 and PP2A^44,45,46.
50 µg
5 x 50 µg


TYROSINE PHOSPHATASES

TYROSINE PHOSPHATASE ENZYMES

SE-135
CD45 protein tyrosine phosphatase (human, recombinant)
>90% by SDS-PAGE, 95 kDa, Storage: -70°C
Human CD45 cytoplasmic domain (residues 584-1281), expressed in yeast^47,48. Contains no detectable protease activity. Supplied at 0.2 mg/ml. Specific activity: ~20 kU/mg. One unit will hydrolyse 1 nmol p-nitrophenyl phosphate per minute at pH 7 and 30°C. K_m=5 mM for pNPP. >95% inhibition of phosphatase activity in the presence of 100 µM ammonium molybdate. A highly useful tool to study the regulation of CD45 tyrosine phosphatase; to remove phosphate specifically from tyrosine residues in proteins; and for drug discovery screening of CD45 phosphatase inhibitors.
20 µg
5 x 20 µg

SE-113
LAR protein tyrosine phosphatase (human, recombinant)
Human transmembrane leukocyte antigen related PTP
>95%, 37 kDa, Storage: -20°C
A 350 amino acid, soluble catalytic LAR-D1 domain, expressed in E. coli⁴⁹. Contains no detectable protease, DNase, RNase, or Ser/Thr phosphatase activity. Supplied in 50% glycerol buffer. Specific activity: 4 kU/mg. One unit will hydrolyze 1 nmol p-nitrophenyl phosphate per minute at pH 7.0 and 30°C. 20 U can dephosphorylate >95% of phosphotyrosine in 1 nmol protein in 30 minutes^50,51.
100 U

SE-114
T-cell protein tyrosine phosphatase, (human, recombinant)
>95%, 38 kDa, Storage: -20°C
T-cell phosphatase with a C-terminal 11 kDa deletion (TCDC11), expressed in E. coli^52,53. Contains no detectable protease, DNase, RNase, or Ser/Thr phosphatase activity. Supplied in 50% glycerol with stabilizers. Specific activity: 15 kU/mg. One unit will release 1 nmol p-nitrophenyl phosphate per minute at pH 7 and 30°C. One unit can dephosphorylate >95% of phosphotyrosine in 1 nmol protein in 30 minutes.
100 U

SE-115
Yop protein tyrosine phosphatase (from Yersinia enterocolitica)
>95%, 51 kDa Storage: -20°C
Protein from Yop51* gene that contains the C235R mutation, expressed in E. coli^54,55,56. Contains no detectable protease, DNase, RNase, or Ser/Thr phosphatase activity. Supplied in 50% glycerol with stabilizers. Specific activity: 500 kU/mg. One unit will hydrolyze 1 nmol p-nitrophenyl phosphate per minute at pH 7.0 and 30°C. 1 kU can dephosphorylate >95% of phosphotyrosine in 1 nmol protein in 30 minutes.
20 kU


TYROSINE PHOSPHATASE INHIBITORS

EI-244
Benzylphosphonic acid
98%, MW=172.1 [6881-57-8] Storage: RT
Benzylphosphonic acid is a nonhydrolyzable structural mimic of phosphotyrosine. It has been shown to inhibit a tyrosine-protein phosphatase from bovine heart (K_i=4.6 mM)⁵⁷. Benzylphosphonic acid is not cell permeable.
100 mg
500 mg

EI-245
Benzylphosphonic acid-(AM)₂
98%, MW=316.3, Storage: -20°C
Benzylphosphonic acid-(AM)₂ is a bis-acetoxymethyl ester (AM) derivative of benzylphosphonic acid which is cell permeable and is converted to the active phosphonic acid via intracellular esterase-catalyzed cleavage of the AM esters.
10 mg
50 mg

T-100
L-p-Bromotetramisole oxalate
98%, MW=373.2 [62284-79-1] Storage: RT
L-p-Bromotetramisole is a well known inhibitor of alkaline phosphatases which was recently found to inhibit tyrosine phosphatase. This activity was identified by its ability to mimic the action of orthovanadate (a well known tyrosine phosphatase inhibitor)⁵⁸ in the potentiation of fluorouracil antiproliferative activity⁵⁹.
10 mg
50 mg

T-107
D-p-Bromotetramisole oxalate
99%, MW=373.2 [71461-24-0] Storage: RT
D-p-bromotetramisole is an inactive isomer of bromotetramisole and may be used as a negative control⁵⁹.
10 mg
50 mg

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