Acute serum amyloid A is an endogenous TLR2 ligand that mediates inflammatory and angiogenic mechanisms.

INTRODUCTION: Acute-phase serum amyloid A (A-SAA) has cytokine-like properties and is expressed at sites of inflammation. We examined whether A-SAA-induced pro-inflammatory mechanisms are mediated through Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA). METHODS: The effect of A-SAA on human embryonic kidney (HEK), TLR2 or TLR4 cells was quantified by nuclear factor (NF)-κB luciferase reporter assays. A-SAA-induced RASFC and dHMVEC function were performed in the presence of a specific neutralising anti-TLR2 mAb (OPN301) (1 µg/mL) and matched IgG isotype control Ab (1 µg/mL). Cell surface expression of intracellular adhesion molecule (ICAM)-1, chemokine expression, cell migration, invasion and angiogenesis were assessed by flow cytometry, ELISA, Matrigel invasion chambers and tube formation assays. MyD88 expression was assessed by real-time PCR and western blot. RESULTS: A-SAA induced TLR2 activation through induction of NF-κB (p<0.05), but failed to induce NF-κB in HEK-TLR4 cells, confirming specificity for TLR2. A-SAA-induced proliferation, invasion and migration were significantly inhibited in the presence of anti-TLR2 (all p<0.05), with no significant effect observed for tumour necrosis factor-α-induced events. Additionally, A-SAA-induced ICAM-1, interleukin-8, monocyte chemoattractant protein-1, RANTES and GRO-α expression were significantly reduced in the presence of anti-TLR2 (all p<0.05), as was A-SAA induced angiogenesis (p<0.05). Finally, A-SAA induced MyD88 signalling in RASFC and dHMVEC (p<0.05). CONCLUSIONS: A-SAA is an endogenous ligand for TLR2, inducing pro-inflammatory effects in RA. Blocking the A-SAA/TLR2 interaction may be a potential therapeutic intervention in RA.

The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

Toll-like receptor 7 (TLR7) plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV) and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM) plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM(-/-) murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-ß, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNß, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNß and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM(-/-) cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

WITHDRAWN: Modulation of TLR3, TLR4 and TLR7 mediated IFN-ß, Rantes and TNFα production by HIVEP1.

The manuscript was withdrawn by the author.

TRIF-mediated TLR3 and TLR4 signaling is negatively regulated by ADAM15.

TLRs are a group of pattern-recognition receptors that play a crucial role in danger recognition and induction of the innate immune response against bacterial and viral infections. The TLR adaptor molecule, Toll/IL-1R domain-containing adaptor inducing IFN (TRIF), facilitates TLR3 and TLR4 signaling and concomitant activation of the transcription factors, NF-κB and IFN regulatory factor 3, leading to proinflammatory cytokine production. Whereas numerous studies have been undertaken toward understanding the role of TRIF in TLR signaling, little is known about the signaling components that regulate TRIF-dependent TLR signaling. To this end, TRIF-interacting partners were identified by immunoprecipitation of the TRIF signaling complex, followed by protein identification using liquid chromatography mass spectrometry. Following stimulation of cells with a TLR3 or TLR4 ligand, we identified a disintegrin and metalloprotease (ADAM)15 as a novel TRIF-interacting partner. Toward the functional characterization of the TRIF:ADAM15 interaction, we show that ADAM15 acts as a negative regulator of TRIF-mediated NF-κB and IFN-ß reporter gene activity. Also, suppression of ADAM15 expression enhanced polyriboinosinic polyribocytidylic acid and LPS-mediated proinflammatory cytokine production via TRIF. In addition, suppression of ADAM15 expression enhanced rhinovirus 16 and vesicular stomatitis virus-mediated proinflammatory cytokine production. Interestingly, ADAM15 mediated the proteolytic cleavage of TRIF. Thus, ADAM15 serves to curtail TRIF-dependent TLR3 and TLR4 signaling and, in doing so, protects the host from excessive production of proinflammatory cytokines and matrix metalloproteinases. In conclusion, to our knowledge, our study clearly shows for the first time that ADAM15 plays an unexpected role in TLR signaling, acting as an anti-inflammatory molecule through impairment of TRIF-mediated TLR signaling.

14-3-3ε and 14-3-3σ inhibit Toll-like receptor (TLR)-mediated proinflammatory cytokine induction.

Toll-like receptors (TLRs) are a group of pattern recognition receptors that play a crucial role in the induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral double-stranded RNA. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Limited studies have applied proteomics toward understanding the dynamics of TLR signaling. Herein, a proteomics approach identified 14-3-3ε and 14-3-3σ proteins as new members of the TLR signaling complex. Toward the functional characterization of 14-3-3ε and 14-3-3σ in TLR signaling, we have shown that both of these proteins impair TLR2, TLR3, TLR4, TLR7/8, and TLR9 ligand-induced IL-6, TNFα, and IFN-ß production. We also show that 14-3-3ε and 14-3-3σ impair TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated NF-κB and IFN-ß reporter gene activity. Interestingly, although the 14-3-3 proteins inhibit poly(I:C)-mediated RANTES production, 14-3-3 proteins augment Pam(3)CSK(4), LPS, R848, and CpG-mediated production of RANTES (regulated on activation normal T cell expressed and secreted) in a Mal (MyD88 adaptor-like)/MyD88-dependent manner. 14-3-3ε and 14-3-3σ also bind to the TLR adaptors and to both TRAF3 and TRAF6. Our study conclusively shows that 14-3-3ε and 14-3-3σ play a major regulatory role in balancing the host inflammatory response to viral and bacterial infections through modulation of the TLR signaling pathway. Thus, manipulation of 14-3-3 proteins may represent novel therapeutic targets for inflammatory conditions and infections.

Cancer and viruses: a double-edged sword.

Oncovirus, synonymously called a 'tumour virus', is a virus that can cause cancer. An oncolytic virus preferentially infects the host's cancer cells and lyses them, causing tumour destruction, and is thus referred to as a 'cancer killing virus'. With an estimated 11% of cancer-associated deaths caused by oncoviruses and the possibility that many cancers may be treated by using oncolytic viruses, the role of viruses in cancer may be viewed as a double-edged sword. A total of seven human cancer viruses have been identified as oncoviruses, having been associated with various cancers. Conversely, a large number of oncolytic viruses have shown great potential towards the treatment of certain types of cancer. Proteomics has now been applied towards understanding the complex interplay that exists between oncoviruses and the immune responses that serve to prevent oncoviral diseases. This review attempts to summarise the neoplastic potential of human tumour associated viruses and associated vaccine successes. The potential use of oncolytic viruses for the therapeutic intervention of cancer will also be discussed. Finally, this review will discuss the enormous potential of proteomics technology in the field of oncovirology.

Nuclear factor κB subunits RelB and cRel negatively regulate Toll-like receptor 3-mediated ß-interferon production via induction of transcriptional repressor protein YY1.

The induction of ß-interferon (IFN-ß) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-ß requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-ß promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-ß promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-ß. Herein, we examined the role of NF-κB in regulating IFN-ß expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-ß expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-ß expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-ß promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-ß expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-ß expression.

Targeted disruption of nonribosomal peptide synthetase pes3 augments the virulence of Aspergillus fumigatus.

Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes3 (AFUA_5G12730), by targeted gene deletion and extensive phenotypic analysis. It was observed that in contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P < 0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion of pes3 also resulted in increased susceptibility to the antifungal, voriconazole (P < 0.01), shorter germlings, and significantly reduced surface ß-glucan (P = 0.0325). Extensive metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus. Proteome alterations in A. fumigatus Δpes3 strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase.

Absence of MyD88 results in enhanced TLR3-dependent phosphorylation of IRF3 and increased IFN-ß and RANTES production.

Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in "danger" recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN-ß. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN-ß induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF-α induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN-ß and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKKε-, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN-ß induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN-ß. Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKKε-mediated induction of IRF3 and concomitant IFN-ß and RANTES production.

TLR3-mediated IFN-ß gene induction is negatively regulated by the TLR adaptor MyD88 adaptor-like.

There is limited insight into the mechanisms involved in the counterregulation of TLR. Given the important role of TLR3/TIR domain-containing adaptor-inducing IFN-ß (TRIF)-dependent signalling in innate immunity, novel insights into its modulation is of significance in the context of many physiological and pathological processes. Herein, we sought to perform analysis to definitively assign a mechanistic role for MyD88 adaptor-like (Mal), an activator of TLR2/4 signalling, in the negative regulation of TLR3/TRIF signalling. Biochemical and functional analysis demonstrates that Mal negatively regulates TLR3, but not TLR4, mediated IFN-ß production. Co-immunoprecipitation experiments demonstrate that Mal associates with IRF7 (IRF, IFN regulatory factor), not IRF3, and Mal specifically blocks IRF7 activation. In doing so, Mal impedes TLR3 ligand-induced IFN-ß induction. Interestingly, Mal does not affect the induction of IL-6 and TNF-α upon TLR3 ligand engagement. Together, these data show that the TLR adaptor Mal interacts with IRF7 and, in doing so, impairs IFN-ß induction through the positive regulatory domains I-III enhancer element of the IFN-ß gene following poly(I:C) stimulation. Our findings offer a new mechanistic insight into TLR3/TRIF signalling through a hitherto unknown mechanism whereby Mal inhibits poly(I:C)-induced IRF7 activation and concomitant IFN-ß production. Thus, Mal is essential in restricting TLR3 signalling thereby protecting the host from unwanted immunopathologies associated with excessive IFN-ß production.

Expression analysis of the Toll-like receptors in human peripheral blood mononuclear cells.

Toll-like receptors (TLRs) are key regulators of the innate and adaptive immune response to bacterial, viral, and fungal pathogens. To date, 10 human TLRs and 13 mouse TLRs have been identified and they exhibit tissue-specific mRNA/protein expression patterns. Thus, it is essential that the TLR expression profile of model cell lines be delineated prior to experimentation in order to establish whether the requisite TLRs are expressed in the cell line/type of interest. This may be quickly achieved by employing a reverse transcription-polymerase chain reaction (RT-PCR) approach whereby total RNA isolated from the cell type of interest is used as a template for RT-PCR analysis of TLR expression using TLR1-TLR10 specific oligonucleotides. Herein, total RNA was isolated from human peripheral blood mononuclear cells (PBMCs) and its integrity was confirmed by formaldehyde-formamide RNA gel electrophoresis. Thereafter, total RNA was used as a template for RT-PCR analysis using oligonucleotides specific for the amplification of TLR1-10. We have shown that PBMCs express mRNA encoding TLR1-10. These findings suggest that PBMCs may represent a useful TLR-responsive model cell line for examining TLR1-10 signalling events.

NF-kappaB activation by the Toll-IL-1 receptor domain protein MyD88 adapter-like is regulated by caspase-1.

Toll-like receptors (TLRs)-2 and -4 are important proteins in innate immunity, recognizing microbial products and eliciting host defense responses. Both use the adapter proteins MyD88 and MyD88 adapter-like (Mal) to activate signaling pathways. Here we report that Mal but not MyD88 interacts with caspase-1, the enzyme that processes the precursors of the proinflammatory cytokines IL-1beta and IL-18. The interaction was found in a yeast two-hybrid screen and was confirmed by reciprocal GST pull-downs and coimmunoprecipitation of endogenous proteins. We were unable to implicate Mal in regulating caspase-1 activation. However, we found that Mal was cleaved by caspase-1 and that inhibition of caspase-1 activity blocked TLR2- and TLR4-mediated NF-kappaB and p38 MAP kinase activation but not IL-1 or TLR7 signaling, which are Mal independent. These responses, and the induction of TNF, were also attenuated in caspase-1-deficient cells. Finally, unlike wild-type Mal, a mutant Mal, which was not cleaved by caspase-1, was unable to signal and acted as a dominant negative inhibitor of TLR2 and TLR4 signaling. Our study therefore reveals a role for caspase-1 in the regulation of TLR2 and TLR4 signaling pathways via an effect on Mal. This functional interaction reveals an important aspect of the coordination between TLRs and caspase-1 during the innate response to pathogens.

New insights into the regulation of TLR signaling.

Toll-like receptor (TLR) activation is dictated by a number of factors including the ligand itself and the localization of the receptor, in terms of expression profile and subcellular localization and the signal transduction pathway that has been activated. Recent work into TLR signal transduction has revealed complex regulation at a number of different levels including regulation by phosphorylation, targeted degradation, and sequestration of signaling molecules. Here, we describe recent advances that have been made in our understanding of how TLR signaling is regulated at the biochemical level.

Interferon regulatory factor-3-mediated activation of the interferon-sensitive response element by Toll-like receptor (TLR) 4 but not TLR3 requires the p65 subunit of NF-kappa.

Interferon regulatory factor (IRF) 3 is a transcription factor that binds the interferon-sensitive response element (ISRE) and is activated by Toll-like receptor 3 (TLR3) and TLR4. We have found that a dominant negative form of I kappa B kinase 2 and a mutant form of I kappa B, which acts as a super-repressor of NF-kappa B, blocked activation of the ISRE by the TLR4 ligand lipopolysaccharide but not the TLR3 ligand poly(I-C). TLR4 failed to activate the ISRE in mouse embryonic fibroblasts bearing a targeted deletion of p65, whereas the response to TLR3 in these cells was normal. The p65 subunit of NF-kappa B was detected in the lipopolysaccharide-activated but not poly(I-C)-activated ISRE-binding complex. Finally, p65 promoted transactivation of gene expression by IRF-3. These results therefore indicate that IRF-3-mediated activation of the ISRE by TLR4 but not TLR3 requires the p65 subunit of NF-kappa B.

Palmitoylation of the human prostacyclin receptor. Functional implications of palmitoylation and isoprenylation.

We have previously established that isoprenylation of the prostacyclin receptor (IP) is required for its efficient G protein coupling and effector signaling (Hayes, J. S., Lawler, O. A., Walsh, M. T., and Kinsella, B. T. (1999) J. Biol. Chem. 274, 23707-23718). In the present study, we sought to investigate whether the IP may actually be subject to palmitoylation in addition to isoprenylation and to establish the functional significance thereof. The human (h) IP was efficiently palmitoylated at Cys(308) and Cys(311), proximal to transmembrane domain 7 within its carboxyl-terminal (C)-tail domain, whereas Cys(309) was not palmitoylated. The isoprenylation-defective hIP(SSLC) underwent palmitoylation but did not efficiently couple to G(s) or G(q), confirming that isoprenylation is required for G protein coupling. Deletion of C-tail sequences distal to Val(307) generated hIP(Delta307) that was neither palmitoylated nor isoprenylated and did not efficiently couple to G(s) or to G(q), whereas hIP(Delta312) was palmitoylated and ably coupled to both effector systems. Conversion of Cys(308), Cys(309), Cys(311), Cys(308,309), or Cys(309,311) to corresponding Ser residues, while leaving the isoprenylation CAAX motif intact, did not affect hIP coupling to G(s) signaling, whereas mutation of Cys(308,311) and Cys(308,309,311) abolished signaling, indicating that palmitoylation of either Cys(308) or Cys(311) is sufficient to maintain functional G(s) coupling. Although mutation of Cys(309) and Cys(311) did not affect hIP-mediated G(q) coupling, mutation of Cys(308) abolished signaling, indicating a specific requirement for palmitoylation of Cys(308) for G(q) coupling. Consistent with this, neither hIP(C308S,C309S), hIP(C308S,C311S), nor hIP(C308S,C309S,C311S) coupled to G(q). Taken together, these data confirm that the hIP is isoprenylated and palmitoylated, and collectively these modifications modulate its G protein coupling and effector signaling. We propose that through lipid modification followed by membrane insertion, the C-tail domain of the IP may contain a double loop structure anchored by the dynamically regulated palmitoyl groups proximal to transmembrane domain 7 and by a distal farnesyl isoprenoid permanently attached to its carboxyl terminus.

Characterization of the 5' untranslated region of alpha and beta isoforms of the human thromboxane A2 receptor (TP). Differential promoter utilization by the TP isoforms.

In humans, thromboxane (TX) A2 signals through two TXA2 receptor (TP) isoforms, TPalpha and TPbeta, that diverge within their carboxyl terminal cytoplasmic (C) tail regions and arise by differential splicing. The human TP gene contains three exons E1-E3; while E1 exclusively encodes 5' untranslated region (UTR) sequence, E2 and E3 represent the main coding exons. An additional noncoding exon, E1b was identified within intron 1. Additionally, the TP gene contains two promoters P1 and P2 located 5' of E1 and E1b, respectively. Herein, we investigated the molecular basis of the differential expression of the TP isoforms by characterizing the 5' UTR of the TP transcripts. While E1 and E1b were found associated with TP transcript(s), their expression was mutually exclusive. 5' rapid amplification of cDNA ends (5' RACE) established that the major transcription initiation (TI) sites were clustered between -115 and -92 within E1 and at -99 within E1b. While E1 and E1b sequences were identified on TPalpha transcript(s), neither existed on TPbeta transcript(s). More specifically, TPalpha and TPbeta transcripts diverged within E2 and the major TI sites for TPbeta transcripts mapped to -12/-15 therein. Through genetic reporter assays, a previously unrecognized promoter, termed P3, was identified on the TP gene located immediately 5' of -12. The proximity of P3 to the TI site of TPbeta suggests a role for P3 in the control of TPbeta expression and implies that TPalpha and TPbeta, in addition to being products of differential splicing, are under the transcriptional control of distinct promoters.

Investigation of the mechanisms of G protein: effector coupling by the human and mouse prostacyclin receptors. Identification of critical species-dependent differences.

We recently identified a novel mechanism explaining how the mouse (m) prostacyclin receptor (IP) couples to Galpha(s), Galpha(i), and Galpha(q) (Lawler, O. A., Miggin, S. M., and Kinsella, B. T. (2001) J. Biol. Chem. 276, 33596-33607) whereby mIP coupling to Galpha(i) and Galpha(q) is dependent on its initial coupling to Galpha(s) and subsequent phosphorylation by cAMP-dependent protein kinase A (PKA) on Ser(357). In the current study, the generality of that mechanism was investigated by examining the G protein coupling specificity of the human (h) IP. The hIP efficiently coupled to Galpha(s)/adenylyl cyclase and to Galpha(q)/phospholipase C activation but failed to couple to Galpha(i). Coupling of the hIP to Galpha(q), or indeed to Galpha(s) or Galpha(i), was unaffected by the PKA or protein kinase C (PKC) inhibitors H-89 and GF 109203X, respectively. Thus, mIP and hIP exhibit essential differences in their coupling to Galpha(i) and in their dependence on PKA in regulating their coupling to Galpha(q). Analysis of their primary sequences revealed that the critical PKA phosphorylation site within the mIP, at Ser(357), is replaced by a PKC site within the hIP, at Ser(328). Conversion of the PKC site of the hIP to a PKA site generated hIP(QL325,326RP) that efficiently coupled to Galpha(s) and to Galpha(i) and Galpha(q); coupling of hIP(QL325,326RP) to Galpha(i) but not to Galpha(s) or Galpha(q) was inhibited by H-89. Abolition of the PKC site of the hIP generated hIP(S328A) that efficiently coupled to Galpha(s) and Galpha(q) but failed to couple to Galpha(i). Finally, conversion of the PKA site at Ser(357) within the mIP to a PKC site generated mIP(RP354,355QL) that efficiently coupled to Galpha(s) but not to Galpha(i) or Galpha(q). Collectively, our data highlight critical differences in signaling by the mIP and hIP that are regulated by their differential phosphorylation by PKA and PKC together with contextual sequence differences surrounding those sites.