IP3 (inositol 1,4,5-trisphosphate) receptors form tetrameric, IP3-gated Ca2+ channels in endoplasmic reticulum membranes, and are substrates for several kinases, including PKA (cAMP-dependent protein kinase). Activation of PKA has been reported to either enhance or inhibit type III IP3 receptor Ca2+-channel activity, but, as yet, the sites of phosphorylation remain unknown. Here, we reveal that PKA phosphorylates the type III IP3 receptor at Ser916, Ser934 and Ser1832, and that, intriguingly, each site is located close to a putative surface-exposed peptide loop. Furthermore, we demonstrate that Ser934 is considerably more susceptible to PKA-dependent phoshorylation than either Ser916 or Ser1832. These findings define the sites at which the type III IP3 receptor is phosphorylated by PKA, and provide the basis for exploring the functional consequences of this regulatory event.

g Noonan syndrome (NS) is an autosomal dominant disorder caused by activating mutations in the PTPN11 gene encoding Shp2, which manifests in congenital heart disease, short stature, and facial dysmorphia. The complexity of Shp2 signaling is exemplified by the observation that LEOPARD syndrome (LS) patients possess inactivating PTPN11 mutations yet exhibit similar symptoms to NS. Here, we identify "protein zero-related" (PZR), a transmembrane glycoprotein that interfaces with the extracellular matrix to promote cell migration, as a major hyper-tyrosyl-phosphorylated protein in mouse and zebrafish models of NS and LS. PZR hyper-tyrosyl phosphorylation is facilitated in a phosphatase-independent manner by enhanced Src recruitment to NS and LS Shp2. In zebrafish, PZR overexpression recapitulated NS and LS phenotypes. PZR was required for zebrafish gastrulation in a manner dependent upon PZR tyrosyl phosphorylation. Hence, we identify PZR as an NS and LS target. Enhanced PZR-mediated membrane recruitment of Shp2 serves as a common mechanism to direct overlapping pathophysiological characteristics of these PTPN11 mutations.

Intracellular signaling pathways that are mediated by tyrosyl phosphorylation are controlled through the balanced and opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The view that PTPs are equal contributing partners in the regulation of cellular tyrosyl phosphorylation continues to mature. However, there still remains the perception that PTPs play largely housekeeping roles. A growing body of evidence firmly dispels this perception, and it is evident that PTPs function with stringent signaling specificity, in concert with their PTK counterparts, to define specific biological outcomes. In this review, we will focus on illustrating that PTPs exhibit defined substrate specificity and subsequently regulate signaling pathways in a precise manner. The PTP SuperfamilyThe superfamily of PTPs is characterized by a consensus signature motif represented by HC(X) 5 R, which defines the active site of these enzymes. Two classes of PTPs can be defined (FIGURE 1). The first class is composed of classical PTPs that are defined by cysteine-based phosphotyrosine specificity, and the second class constitutes the dual-specificity phosphatases (DSPs). There are 37 classical human PTP genes that contain at least one PTP domain of ~280 amino acids. The DSPs are much larger in number, and there are 65 of these genes in the human genome. The classical PTP genes can be further subdivided into transmembrane receptor PTPs (RPTPs), or non-transmembrane PTPs, which localize to the cytoplasm (2, 4, 68). There are 12 RPTPs containing tandem PTP domains with the remainder containing a single PTP domain. The membrane-proximal PTP domain (D1) and membrane-distal PTP domain (D2) serve distinct roles. In the majority of cases, the D1 PTP domain comprises the active domain, whereas in most cases, but not all, the D2 domain serves a negative regulatory role. Although still quite controversial, evidence has been put forth to suggest that RPTPs are regulated through dimerization whereby RPTP activity is inhibited upon dimerization. However, there is also evidence against such a model of RPTP regulation, and further work in this particular area is warranted. Much like the RTKs, RPTPs utilize their diverse extracellular domains to transduce intracellular signals through binding to soluble ligands, but, in addition, RPTPs utilize their extracellular domains to mediate cell-cell and cell-matrix interactions (33,35,45,73). The nontransmembrane or cytoplasmic PTPs contain a single PTP domain, and their diversity is derived through noncatalytic regulatory domains that reside either at the NH 2 or COOH terminus of the PTP domain (2-3, 68). The noncatalytic domains are critical for exerting PTP substrate specificity and include one or more of the following features: 1) regulation of PTP activity, 2) directing subcellular localization, and 3) targeting PTP protein-protein interactions. Hence, the noncatalytic regions of the non-transmembrane PTPs offer a broad level of structural diversity, and together with the PTP...

Type I inositol 1,4,5-trisphosphate receptors can be phosphorylated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). To define the site-specificity of these events we analyzed the phosphorylation of mutant receptors expressed in intact cells. These studies showed that S(1588) and S(1755), the serine residues within kinase consensus sequences, are equally sensitive to PKA, that phosphorylation events at these sites are independent of each other, and that PKG predominantly phosphorylates S(1588). These findings provide the basis for understanding the functional consequences of type I inositol 1,4,5-trisphosphate receptor phosphorylation.

Several studies have shown that PKA-mediated phosphorylation of IP 3 R1 at serines S 1588 and S 1755 enhances the receptor's ability to mobilize Ca 2+ . In contrast, much less is known about whether Ca 2+ mobilization via IP 3 R2 and IP 3 R3 is regulated by PKA. We report here that IP 3 R2 is only very weakly phosphorylated in response to PKA activation and is probably not a physiological substrate for this kinase. IP 3 R3, however, is known to be phosphorylated by PKA at three sites (S 916 , S 934 and S 1832 ) and, thus, we examined how phosphorylation of these sites affects Ca 2+ mobilization in DT40-3KO cells stably expressing either exogenous wild-type or mutant IP 3 R3s; an antibody raised against phospho-serine 934 of IP 3 R3 was used to demonstrate that the exogenous IP 3 R3s are strongly phosphorylated in response to PKA activation. Surprisingly, our data show that IP 3 R3-mediated Ca 2+ mobilization is unaffected by phosphorylation of S 916 , S 934 and S 1832 . In contrast, phosphorylation of exogenous IP 3 R1 (monitored with an antibody against phospho-serine 1755) enhances Ca 2+ mobilization, indicating that DT40-3KO cells have the capacity to respond to phosphorylation of IP 3 Rs. Overall, these data suggest that modification of Ca 2+ flux may not be the primary effect of IP 3 R3 phosphorylation by PKA.