Insulin and growth factors stimulate the phosphorylation of a Mr-22000 protein in 3T3-L1 adipocytes

Abstract

Insulin, epidermal growth factor (EGF), platelet-derived growth factor, multiplication-stimulating activity and 10% foetal-calf serum each stimulated the phosphorylation of a cytosolic Mr-22000 acidic heat-stable protein in Swiss mouse 3T3-L1 adipocytes. Phosphorylation of this protein was not stimulated by isoprenaline or dibutyryl cyclic AMP. The effect of insulin was maximal (3-fold increase) by 10 min; half-maximal stimulation was observed at 70 pM-insulin. Both [32P]phosphoserine and [32P]phosphothreonine residues were present in the Mr-22000 protein after insulin- and growth-factor-stimulated phosphorylation, but no [32P]phosphotyrosine. The major site of insulin- and EGF-stimulated phosphorylation appeared to be a threonine residue, in contrast with previously studied insulin-stimulated phosphorylation of serine residues. Insulin treatment appeared to result in a shift of the protein toward the anode on isoelectric focusing. Insulin and EGF present simultaneously did not lead to phosphorylation beyond that seen with each hormone singly. We surmise that insulin, EGF and perhaps other growth factors may activate a common protein kinase or inhibit a common protein phosphatase in 3T3-L1 adipocytes which acts on the Mr-22000 protein.

Phosphorylation of the eIF4E‐binding protein PHAS‐I after exposure of PC12 cells to EGF and NGF

“…The inability of alkaline phosphatase to convert the ~-form may be due to inaccessibility of the phosphate residue or a difference in amino acid residues that are dephosphorylated. It has been shown that PHAS-I can be phosphorylated at serine as well as threonine residues [10]. Second, azp labeling of cells showed two phoshorylated PHAS-I forms [4,9,30], and their migration on SDS-PAGE indicates that these phosphorylated forms are the [3-and y-form.…”

“…It has been shown that PHAS-I is mostly phosphorylated at serine residues in control cells, but at threonine residues in insulin-treated cells [10]. This suggests that more than one kinase is involved in PHAS-I phosphorylation.…”

“…Studies with 32p labeling and two-dimensional separation resulted in identification of 2 labeled forms of PHAS-I, in contrast to the 14 spots identified by immunoblotting [10,12]. To establish the relationship between the c~-, [3-, and y-forms, extracts of PC12 cells were treated with alkaline phosphatase.…”

“…The ¢x and 13 forms of PHAS-I are able to complex with eIF4E [4,5], thereby impairing the association of eIF4E with eIF4G, necessary for initiation. Phosphorylation of PHAS-I is increased after treatment of cells with insulin, epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) or platelet-derived growth factor (PDGF) [10][11][12]. The signal transduction pathway leading to PHAS-I phosphorylation and the in vivo kinase(s) of PHAS-I remain unclear.…”

Control of PHAS-I phosphorylation in 3T3-L1 adipocytes: effects of inhibiting protein phosphatases and the p70 S6K signalling pathway

“…Regardless of the mechanism, to the extent that eIF-4E is involved in the control of mitogenesis, PHAS-I is also implicated as it is a regulator of eIF-4E. The phosphorylation of PHAS-I and release of eIF-4E are stimulated by several mitogens, including insulinlike growth factor-1, platelet-derived growth factor BB, and epidermal growth factor [11,41]. As suggested previously [11,12], the dephosphorylation of PHAS-I produced by rapamycin could be involved in the antiproliferative effects of the agent.…”