The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the "negative" regulation of cell signaling. The molecular basis for the regulation of SHP-1 is incompletely understood. Whereas SHP-1 has previously been shown to be phosphorylated on two tail tyrosine residues (Tyr 536 and Tyr 564 ) by several protein-tyrosine kinases, the effects of these phosphorylation events have been difficult to address because of the intrinsic instability of the linkages within a protein-tyrosine phosphatase. Using expressed protein ligation, we have generated semisynthetic SHP-1 proteins containing phosphotyrosine mimetics at the Tyr 536 and Tyr 564 sites. Two phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F 2 Pmp). Incorporation of Pmp at the 536 site led to 4-fold stimulation of the SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect. Incorporation of F 2 Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity and 1.6-fold at the 564 site. A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition. In contrast, tyrosine phosphorylation at the 564 site has the potential to engage the C-Src homology 2 domain intramolecularly, which can modestly and indirectly influence catalytic activity. The finding that phosphonate modification at each of the 536 and 564 sites can promote interaction with the Grb2 adaptor protein indicates that the intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.

Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain) produces total inhibition of ATP utilization and enzyme phosphorylation by P(i), without a significant effect on Ca(2+) binding.

High-affinity and cooperative binding of two Ca(2+) per ATPase (SERCA) occurs within the membrane-bound region of the enzyme. Direct measurements of binding at various Ca(2+) concentrations demonstrate that site-directed mutations within this region interfere selectively with Ca(2+) occupancy of either one or both binding sites and with the cooperative character of the binding isotherms. A transition associated with high affinity and cooperative binding of the second Ca(2+) and the engagement of N796 and E309 are both required to form a phosphoenzyme intermediate with ATP in the forward direction of the cycle and also to form ATP from phosphoenzyme intermediate and ADP in the reverse direction of the cycle. This transition, defined by equilibrium and kinetic characterization of the partial reactions of the enzyme cycle, extends from transmembrane helices to the catalytic site through a long-range linkage and is the mechanistic device for interconversion of binding and phosphorylation potentials.

, and the cycle is then completed by hydrolytic cleavage of the phosphoenzyme. The SR ATPase contains 994 amino acids (4), folded to form 10 transmembrane segments (M1-M10) and a large extramembranous (cytosolic) region (5, 6). Mutational (7) and structural studies (8) have shown that the Ca 2ϩ binding domain resides within the membrane-bound region of the ATPase, at a 50-Å distance from the phosphorylation domain in the cytosolic region of the enzyme. Functional interdependence of Ca 2ϩ and phosphorylation domains occurs through long range intramolecular linkage (9).The cytosolic region of the ATPase includes a short N-terminal segment (Met 1 -Glu 58 ) and the loop (Trp 107 -Ser 261 ) between the M2 and M3 transmembrane segments, folded in the A domain. Furthermore, the cytosolic region includes the large loop between the M4 and M5 transmembrane segments, folded in separate P (phosphorylation) and N (nucleotide binding) domains (8). Although the loops between the remaining transmembrane segments are relatively small, attention has been brought to L67 (Phe 809 -Gly 831 ) by Falson et al. (10) and Menguy et al. (11), who found that cluster mutations of aspartic residues to alanine raise the Ca 2ϩ concentration required for ATPase activation. Our interest on L67 was heightened by its close relationship with the P domain (8), and the major role played by the contiguous M6 segment in Ca 2ϩ binding (12). Therefore, we performed a detailed mutational analysis of L67. MATERIALS AND METHODS DNA Constructs and Vectors-The chicken fast muscle SR ATPase (SERCA-1) cDNA (13) was inserted into the pUC19 plasmid for amplification, and then subcloned into the pSELECT-1 vector for site directed mutagenesis. Mutations were carried out by the Altered Sites in vitro mutagenesis system made available by Promega (Madison, WI), or by overlap extension using the polymerase chain reaction.Wild type and mutated cDNA was subcloned into the shuttle plasmid p⌬E1sp1A (Microbix BioSystems). In the final constructs, the cDNA was preceded by the SV40 or the cytomegalovirus promoter, and followed by the SV40 polyadenylation signal. The shuttle plasmids were either used directly for transfection of COS-1 cells by the DEAE-dextran method, or for cotransfection of HEK293 cells in conjunction with the replication defective adenovirus plasmid pJM17 (Microbix BioSystems) to obtain recombinant adenovirus vectors (14). Alternatively, cDNA constructs were subcloned into pAd-lox shuttle vector and cotransfected with purified ⌿5 adenovirus genome in CRE8 cells derived from the HEK293 line. CRE8 cells constitutively express the Cre recombinase, which catalyzes efficient recombination between loxP sites in the ⌿5 genome and in the pAd-lox to yield recombinant adenovirus *