Therapeutic antibodies in stroke.

Immunotherapy represents an active area of biomedical research to treat cancer, autoimmune diseases, and neurodegenerative disorders. In stroke, recanalization therapy is effective in reducing brain tissue damage after acute ischemic stroke. However, the narrow time window restricts its application for the majority of stroke patients. There is an urgent need to develop adjuvant therapies such as immunotherapy, stem cell replacement, and neuroprotective drugs. A number of molecules have been targeted for immunotherapy in stroke management, including myelin-associated proteins and their receptors, N-methyl-d-aspartic acid receptors, cytokines, and cell adhesion molecules. Both active vaccination and passive antibodies were tested in animal models of acute ischemic stroke. However, the mechanisms underlying the efficacy of immunotherapy are different for each target protein. Blocking myelin-associated proteins may enhance neuroplasticity, whereas blocking adhesion molecules may yield neuroprotection by suppressing the immune response after stroke. Although results from animal studies are encouraging, clinical trials using therapeutic antibodies failed to improve stroke outcome due to severe side effects. It remains a challenge to generate specific therapeutic antibodies with minimal side effects on other organs and systems.

Sprouty2 interacts with protein kinase C delta and disrupts phosphorylation of protein kinase D1.

The Sprouty (Spry) proteins act as inhibitors of the Ras/ERK pathway downstream of receptor tyrosine kinases. In this study, we report a novel interaction between protein kinase C delta (PKCdelta) and Spry2. Endogenous PKCdelta and Spry2 interact in cells upon basic fibroblast growth factor stimulation, indicating a physiological relevance for the interaction. This interaction appeared to require the full-length Spry2 protein and was conformation-dependent. Conformational constraints were released upon FGFR1 activation, allowing the interaction to occur. Although this interaction did not affect the phosphorylation of PKCdelta by another kinase, it reduced the phosphorylation of a PKCdelta substrate, protein kinase D1 (PKD1). Spry2 was found to interact more strongly with PKCdelta with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKCdelta, it was more likely to form a trimeric complex with both PKCdelta and PKD1. Formation of the complex was found to be dependent on an existing PKCdelta-PKD1 interaction. By disrupting the interaction between PKCdelta and PKD1, Spry2 was unable to associate with PKCdelta to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKCdelta and PKD1 was increased, and the transfer of phosphate groups from PKCdelta to PKD1 was at least partly blocked by Spry2. The action of Spry2 on PKCdelta resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKCdelta signaling. By disrupting the capacity of PKCdelta to phosphorylate its cognate substrates, Spry2 may serve to modulate PKCdelta signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome.

Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling.

The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of Spry2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by Spry2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of Spry2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of Spry2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of Spry2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.

Direct binding of PP2A to Sprouty2 and phosphorylation changes are a prerequisite for ERK inhibition downstream of fibroblast growth factor receptor stimulation.

In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (Spry2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of Spry2 is revealed upon stimulation. The activation of Spry2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The band-shifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to Spry2 around residues 50-60. We show that human Spry2 co-immunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to Spry2, but not to Spry2Delta50-60 (Delta50-60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression. c-Cbl and PP2A-A compete for binding centered around Tyr-55 on Spry2. We show that there are at least two distinct pools of Spry2, one that binds PP2A and another that binds c-Cbl. c-Cbl binding likely targets Spry2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for Spry2 to act as a Ras/ERK pathway inhibitor in FGF signaling.

A Src homology 3-binding sequence on the C terminus of Sprouty2 is necessary for inhibition of the Ras/ERK pathway downstream of fibroblast growth factor receptor stimulation.

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.