Phosphatidylinositol 3'-kinase (PI 3'-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We ...have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3'-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3'-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3'-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3'-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3'-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3'-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3'-kinase by tyrosylphosphorylated proteins requires occupancy of both SH2 domains in p85.
Phosphatidylinositol 3â²-kinase (PI 3â²-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in
its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). ...We have previously shown that both tyrosyl-phosphorylated
IRS-1 and mono-phosphopeptides containing a single Y XX M motif activate PI 3â²-kinase in vitro . However, activation by the mono-phosphopeptides was significantly less potent than activation by the multiply phosphorylated
IRS-1. We now show that the increased potency of PI 3â²-kinase activation by IRS-1 relative to phosphopeptide is not due to
tertiary structural features IRS-1, as PI 3â²-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1.
Furthermore, activation of PI 3â²-kinase by bis-phosphorylated peptides containing two Y XX M motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data
suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However,
these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed
to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities,
we produced recombinant PI 3â²-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both
SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3â²-kinase
activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the
first direct evidence that full activation of PI 3â²-kinase by tyrosyl-phosphorylated proteins requires occupancy of both SH2
domains in p85.
1
Many studies indicate an involvement of substance P in the transmission of nociceptive stimuli, without, however, presenting any conclusive evidence as to its exact site and mode of action. The ...present experiments tested the involvement of substance P in the mediation of chemical nociception using the non‐peptidic specific tachykinin NK1 ‐receptor antagonist, RP 67580 (2‐1‐imino‐2‐(2‐methoxyphenyl‐ethyl‐7, 7diphenyl‐4‐perhydroisoindolone (3aR, 7aR)).
2
Mean arterial pressure (MAP) and intragastric pressure (IGP) were measured in anaesthetized rats. The reflex changes of these parameters in response to i.p. or s.c. injections of hydrochloric acid or capsaicin were taken to indicate nociception.
3
Intravenous administration of RP 67580 up to 5 mg kg−1 had little influence on the reflex changes in MAP or IGP in response to hydrochloric acid or capsaicin. In contrast, the sensitization of rats to i.p. capsaicin by preinjection of prostaglandin E2 was significantly reduced by 1 mg kg−1 RP 67580.
4
Intrathecal injection of 5 μg RP 67580 inhibited the reflex changes of MAP and IGP in response to i.p. or s.c. capsaicin whereas the inactive enantiomer RP 68651 was ineffective.
5
The results indicate that spinal NK1 ‐receptors are involved in the acute transmission of chemically induced pain, while such receptors in the periphery take part in the sensitization by prostaglandin E2. The rather minor ability of i.v. RP 67580 to inhibit the acute nociceptive reflex is attributed to an insufficient penetration of the blood‐brain‐barrier.
Many studies indicate an involvement of substance P in the transmission of nociceptive stimuli, without, however, presenting any conclusive evidence as to its exact site and mode of action. The ...present experiments tested the involvement of substance P in the mediation of chemical nociception using the non‐peptidic specific tachykinin NK
1
‐receptor antagonist, RP 67580 (2‐1‐imino‐2‐(2‐methoxyphenyl‐ethyl‐7, 7diphenyl‐4‐perhydroisoindolone (3a
R
, 7a
R
)).
Mean arterial pressure (MAP) and intragastric pressure (IGP) were measured in anaesthetized rats. The reflex changes of these parameters in response to i.p. or s.c. injections of hydrochloric acid or capsaicin were taken to indicate nociception.
Intravenous administration of RP 67580 up to 5 mg kg
−1
had little influence on the reflex changes in MAP or IGP in response to hydrochloric acid or capsaicin. In contrast, the sensitization of rats to i.p. capsaicin by preinjection of prostaglandin E
2
was significantly reduced by 1 mg kg
−1
RP 67580.
Intrathecal injection of 5 μg RP 67580 inhibited the reflex changes of MAP and IGP in response to i.p. or s.c. capsaicin whereas the inactive enantiomer RP 68651 was ineffective.
The results indicate that spinal NK
1
‐receptors are involved in the acute transmission of chemically induced pain, while such receptors in the periphery take part in the sensitization by prostaglandin E
2
. The rather minor ability of i.v. RP 67580 to inhibit the acute nociceptive reflex is attributed to an insufficient penetration of the blood‐brain‐barrier.
PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of ...several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.
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