1. Structure and Mechanism of the Phosphotyrosyl Phosphatase Activator
Yang Chao, Yongna Xing, Yu Chen, Yanhui Xu, Zheng Lin, Zhu Li, Philip D. Jeffrey, Jeffry B. Stock, Yigong Shi 
Molecular Cell 
Volume 23, Issue 4, Pages (August 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Structure of PTPA
(A) Overall structure of human PTPA. The structure can be viewed to contain three subdomains: core (blue), linker (green), and lid (magenta). Secondary structural elements are labeled.
(B) Surface potential of human PTPA. The positively and negatively charged surface areas are colored blue and red, respectively. Note the deep pocket between the core and linker domain. Figures 1B and 3B were prepared using GRASP (Nicholls et al., 1991); all other structural figures were made using MOLSCRIPT (Kraulis, 1991).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Sequence Alignment of PTPA across Species
Conserved amino acids are highlighted in yellow. Secondary structural elements are indicated above the sequences. Color coding of the secondary structural elements is the same as in Figure 1A. Effects of mutation in PP2A binding and ATPase activity are represented by squares and circles, respectively, with the colors indicating the severity of the mutations. Red, loss of function; Orange, partial loss of function; Green, no significant effect. Residues that are close to ATPγS in the crystal structure of ATPγS bound PTPA (Figure 5F) are identified by a thick blue line. The five species are as follows: Homo sapiens (hs), Xenopus laevis (xl), Drosophila melanogaster (dm), Caenorhabditis elegans (ce), Saccharomyces cerevisiae (sc).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Identification of Functional Surface Regions in PTPA
(A) The invariant residues are clustered on one side of the PTPA structure. Among the 68 invariant residues across five species (Figure 2), 39 are fully or partially exposed to solvent. These residues, colored yellow, map to one side of the PTPA structure. In particular, these residues are primarily located in the surface patch that surrounds the deep pocket between the core and the linker domains.
(B) A surface representation of the PTPA structure, with the degree of sequence conservation encoded by the green color. Invariant residues among all five species are colored green; less conserved residues are colored light green.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Identification of the PP2A Binding Surface on the Structure of PTPA
(A) Five missense mutations in PTPA resulted in significantly compromised interaction between PTPA and PP2A A-C dimer. Shown here are representative SDS-PAGE gels stained by Coomassie blue. The interaction was examined using a GST-mediated pull-down assay, with GST-A-C dimer immobilized on glutathione resin and PTPA in the mobile phase. Lanes 13 and 15–18 are colored red to highlight the loss-of-function results for the associated PTPA mutants. Lanes in which no significant loss of binding was observed are colored green.
(B) The five loss-of-function mutations affect amino acids in the same surface area of the PTPA structure. This area surrounds the border between the lid and the linker domain. The residues whose mutation led to compromised interactions with PP2A are highlighted in red. The residues whose mutation did not have a significant impact on interactions with PP2A are shown in green.
(C) A representative native PAGE to illustrate interaction between PTPA and PP2A A-C dimer. Note that the two mutant PTPA proteins migrated faster on native PAGE because of an extra negative charge.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Identification of Amino Acids in PTPA that Are Important for ATPase Activity in the Presence of PP2A
(A) PTPA and PP2A A-C dimer together constitute a composite ATPase. Neither PTPA nor PP2A alone exhibited any detectable ATPase activity. However, incubation of PP2A A-C dimer with PTPA in the presence of ATP/Mg2+ led to a significant ATPase activity. This activity can be inhibited by okadaic acid.
(B) The PTPA-PP2A composite ATPase has an apparent KM value of ∼0.4 mM for ATP. The KM value was determined by using an equimolar amount of PP2A and PTPA.
(C) A number of mutations in PTPA resulted in significantly compromised ATPase activity in the presence of PP2A.
(D) The residues whose mutation led to compromised ATPase activity map to two regions on the PTPA structure. One region (indicated by a magenta oval) coincides with the loss-of-interaction area. The other region (indicated by an orange oval) is in or close to the deep pocket between the core and the linker domains.
(E) Structure of the ATPγS bound PTPA (green and light green) and its comparison with free PTPA (blue and light blue). The only region that undergoes significant change upon binding to ATPγS contains residues 204–210, which are located next to the deep pocket. The five mutations that result in compromised ATPase activity are indicated, and the affected residues are highlighted in red.
(F) Stereo diagram of the bound ATPγS and surrounding region. The 2Fo−Fc electron density map, shown in 1.2 σ, is contoured in gray. The γ-phosphate is flexible and disordered in the crystals and not modeled. Note that five residues whose mutation led to compromised ATPase activity are close to ATPγS. All error bars shown in this figure are standard deviation over three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
PTPA Changes the Phosphatase Specificity of PP2A A-C Dimer from pSer/pThr Substrate to pTyr Substrate
(A) PTPA enhances the pNPPase activity of PP2A A-C dimer in an ATP/Mg2+-dependent manner. Increasing concentrations of PTPA resulted in higher pNPPase activity of PP2A.
(B) ATPγS could not substitute ATP in PTPA-stimulated pNPPase activity of PP2A.
(C) All PTPA mutations that led to compromised ATPase activity also resulted in a compromised ability to stimulate the pNPPase activity of PP2A.
(D) PTPA reduces the pSer/pThr phosphatase activity of PP2A using phosphorylase a as the substrate. Note that wt PTPA and all mutant PTPA that retained binding to PP2A exhibited this activity. PTPA-G290D, which exhibited compromised interaction with PP2A, also lost this activity.
(E) PTPA reduces the pSer/pThr phosphatase activity of PP2A in a concentration-dependent manner. Note that the phosphatase activity reached a maximal reduction at a saturating amount of PTPA and did not exhibit significant decrease beyond this point.
(F) Change of substrate specificity as indicated by relative preference for pNPP over phosphorylase a. These data reflect the ratio of the phosphatase activity for pNPP over that for phosphorylase a.
(G) PTPA enhances the pTyr phosphatase activity of PP2A A-C dimer in an ATP/Mg2+-dependent manner. A pTyr peptide was used as a substrate in these experiments. See Experimental Procedures for details. All PTPA mutations that led to compromised ATPase activity also resulted in a compromised ability to stimulate the pTyr phosphatase activity of PP2A.
(H) A working model of PTPA on the regulation of PP2A function. PTPA is proposed to critically regulate the function of PP2A through alteration of its substrate specificity. In the absence of PTPA or at low concentrations, PP2A A-C dimer is primarily a pSer/pThr phosphatase and readily forms a holoenzyme with a regulatory subunit. In the presence of μM concentrations of PTPA, at least a fraction of PP2A A-C dimer will be bound by PTPA, which leads to a decrease of PP2A's preference for the pSer/pThr substrates. Binding and hydrolysis of ATP further result in an increase of PP2A's preference for the pTyr substrates. This dramatic change of substrate specificity is likely to have a direct consequence for cellular function. All error bars shown in this figure are standard deviation over three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions