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Characterization of growth hormone-induced tyrosine-phosphorylated proteins in mouse cells that express GH receptors
Abstract:
Following the growth hormone (GH) and GH receptor (R) interaction, the receptor and Janus tyrosine kinase 2 (JAK2) become tyrosine phosphorylated along with other intracellular proteins. Previously, we reported that GH induces tyrosine phosphorylation of intracellular proteins with molecular masses of approx 95 kDa (pp95) in mouse 3T3-F442A preadipocytes and in mouse L-cells that express recombinant GHRs. We have studied this GH-induced phosphorylation event in greater detail. Three proteins with apparent molecular masses of 93, 95, and 96 kDa showed increased tyrosine phosphorylation in a time-dependent manner following GH treatment of cells that express GH receptors. GH-induced tyrosine phosphorylation of these proteins is independent of activation of protein kinase C (PKC). Cell fractionation studies revealed that the majority of tyrosine-phosphorylated pp95/96 is located in the cytoplasm. pp95 and pp96 have pIs of approx 6.2. Immunoprecipitation and Western blot analyses revealed that pp93 and pp95/96 are not immunologically related with Stat1, Stat3, Stat4, JAK2, and GHR. Thus, pp93 and pp95/96 may be important GH signal transducers independent of PKC activation and different from the characterized members in the JAK-STAT pathway.
Insights
Growth hormone (GH) triggers tyrosine phosphorylation of specific intracellular proteins, pp93, pp95, and pp96. These proteins are cytoplasmic and distinct from known JAK-STAT pathway members, suggesting novel GH signaling mechanisms.
Area of Science:
- Cellular Biology
- Molecular Endocrinology
- Signal Transduction
Background:
- Growth hormone (GH) initiates cellular responses through its receptor (GHR).
- GH receptor and JAK2 tyrosine phosphorylation are early events in GH signaling.
- Previous work identified GH-induced tyrosine phosphorylation of ~95 kDa proteins (pp95) in specific cell types.
Purpose of the Study:
- To investigate GH-induced tyrosine phosphorylation of intracellular proteins in greater detail.
- To characterize the properties and cellular localization of GH-phosphorylated proteins.
- To determine if these proteins are related to known signaling molecules like JAK-STAT pathway components.
Main Methods:
- Cell culture (3T3-F442A preadipocytes, L-cells expressing GHR).
- Time-dependent analysis of tyrosine phosphorylation following GH treatment.
- Protein kinase C (PKC) activation assays.
- Cell fractionation to determine protein localization.
- Isoelectric focusing (pI) determination.
- Immunoprecipitation and Western blot analysis using antibodies against specific signaling proteins (Stat1, Stat3, Stat4, JAK2, GHR).
Main Results:
- Three proteins (93, 95, and 96 kDa) exhibited time-dependent increases in tyrosine phosphorylation upon GH stimulation.
- GH-induced tyrosine phosphorylation of these proteins was independent of PKC activation.
- The majority of tyrosine-phosphorylated pp95/96 proteins were found in the cytoplasm.
- pp93, pp95, and pp96 were not immunologically related to Stat1, Stat3, Stat4, JAK2, or GHR.
Conclusions:
- GH induces tyrosine phosphorylation of distinct cytoplasmic proteins (pp93, pp95, pp96).
- These novel phosphoproteins are not members of the characterized JAK-STAT pathway.
- pp93 and pp95/96 represent potential novel transducers of GH signaling, independent of PKC activation.