Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.

Abstract

In the present study, insulin is shown to rapidly stimulate by 8‐ to 12‐fold the enzymatic activity of RAC‐PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC‐PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC‐PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin‐induced shift in RAC‐PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin‐stimulated increase in RAC‐PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3‐kinase) in a dose‐dependent manner with a half‐maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC‐PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC‐PK alpha activity can be regulated by the insulin receptor. RAC‐PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.

An inflammatory micro-environment promotes human adipocyte apoptosis

“…In our experiments, phosphorylation of Akt, the key molecule in the insulin signaling pathway (Kohn et al, 1995), was most robustly inhibited by MacCM (Section 3.3.) suggesting that inhibition of Akt is involved in insulin-mediated anti-apoptosis.…”

“…Fat cell apoptosis has been reported in diseases associated with insulin resistance and inflammation including cancer (Prins et al, 1994), HIV-associated lipodystrophy (Domingo et al, 1999) Pharmacological or genetic inhibition of insulin signaling at the level of Akt sensitized human fat cells for apoptosis induction further supporting a causal connection between insulin resistance and apoptosis sensitivity. Akt is a key molecule in the insulin signaling pathway (Kohn et al, 1995). Among the three isoforms of Akt, Akt2 is the most abundant isoform in insulin sensitive tissues (Brazil and Hemmings, 2001) and it is the major isoform responsible for insulin action (Brazil and Hemmings, 2001).…”

Akt activation by growth factors is a multiple-step process: the role of the PH domain

“…(a) Wildtype Akt, E40A, E40C and E40K mutants migrate as two electrophoretically distinct forms, the predominant one being the slow migrating form. In addition to the two electrophoretic forms of Akt detected in serum-starved cells, PDGF stimulation of wildtype Akt-expressing cells gives rise to an even slower migrating Akt form that is due to phosphorylation (Kohn et al, 1995;Burgering and Co er, 1995, also see subsequent experiments) and correlates with increased kinase activity (activation-associated phosphorylation). The stoichiometry of the PDGF-induced phosphorylation is lower than that induced by insulin.…”

“…Akt, one of these kinases, is notable in that it is a direct target of the phosphatidylinositol 3-kinase (PI3-K) Kohn et al, 1995;Andjelkovic et al, 1996;Burgering and Co er, 1995;Klippel et al, 1996;Datta et al, 1996), and as such it plays an important role in regulating many cellular functions that depend on the activity of this enzyme (Kapeller and Cantley, 1994). The activation of Akt by PI3-K was determined by experiments mapping the pathway that regulates Akt following stimulation by a variety of growth factors Kohn et al, 1995;Burgering and Co er, 1995). This was con®rmed by studies showing that the activated catalytic subunit of the PI3-K is su cient to activate Akt in the absence of growth factor stimulation (Klippel et al, 1996;Datta et al, 1996).…”

EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2

“…The two putative Rsk consensus sites within this stretch are boxed in lighter grey and the targeted serine residue shown in bold type. The serine residues identi®ed by Corbalan-Garcia et al (1996b) (Kohn et al, 1995;Marte et al, 1996), but it diplays a phosphorylation consensus sequence reminiscent of that of p90 Rsk-2 (R-X-R-Y-Z-S-Hyd, where X is any amino acid, Y and Z are small amino acids other than glycine and Hyd is a bulky hydrophobic amino acid) (Alessi et al, 1996). However, Akt is unlikely to be a kinase involved in SOS phosphorylation and down regulation since the puri®ed kinase was unable to phosphorylate the two di erent Gst fusions corresponding to the carboxyl terminal region of SOS (data not shown).…”