SOCS/CIS Protein Inhibition of Growth Hormone-stimulated STAT5 Signaling by Multiple Mechanisms*
- From the Department of Biology, Division of Cell and Molecular Biology, Boston University, Boston, Massachusetts 02215
Abstract
The inhibition of growth hormone (GH) signaling by five members of the GH-inducible suppressor of cytokine signaling (SOCS/CIS) family was investigated in transfected COS cells. Complete inhibition of GH activation of the signal transducer STAT5b and STAT5b-dependent transcriptional activity was observed upon expression of SOCS-1 or SOCS-3, while partial inhibition (CIS, SOCS-2) or no inhibition (SOCS-6) was seen with other SOCS/CIS family members. SOCS-1, SOCS-2, SOCS-3, and CIS each strongly inhibited the GH receptor (GHR)-dependent tyrosine phosphorylation of JAK2 seen at low levels of transfected JAK2; however, only SOCS-1 strongly inhibited the GHR-independent tyrosine phosphorylation of JAK2 seen at higher JAK2 levels. To probe for interactions with GHR, in vitrobinding assays were carried out using glutathioneS-transferase-GHR fusion proteins containing variable lengths of GHR's COOH-terminal cytoplasmic domain. CIS and SOCS-2 bound to fusions containing as few as 80 COOH-terminal GHR residues, provided the fusion protein was tyrosine-phosphorylated. By contrast, SOCS-3 binding required tyrosine-phosphorylated GHR membrane-proximal sequences, SOCS-1 binding was tyrosine phosphorylation-independent, and SOCS-6 did not bind the GHR fusion proteins at all. Mutation of GHR's membrane-proximal tyrosine residues 333 and 338 to phenylalanine suppressed the inhibition by SOCS-3, but not by CIS, of GH signaling to STAT5b. SOCS/CIS proteins can thus inhibit GH signaling to STAT5b by three distinct mechanisms, distinguished by their molecular targets within the GHR-JAK2 signaling complex, as exemplified by SOCS-1 (direct JAK2 kinase inhibition), SOCS-3 (inhibition of JAK2 signaling via membrane-proximal GHR tyrosines 333 and 338), and CIS and SOCS-2 (inhibition via membrane-distal tyrosine(s)).
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant DK33765 (to D. J. W.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Biology, 5 Cummington St., Boston, MA 02215. Fax: 617-353-7404; E-mail: djw@bio.bu.edu.
- Abbreviations:
- GH
-
growth hormone
- GHR
-
GH receptor
- JAK2
-
Janus tyrosine kinase 2
- SOCS
-
suppressor of cytokine signaling
- CIS
-
cytokine-inducible SH2 protein
- STAT
-
signal transducer and activator of transcription
- EMSA
-
electrophoretic mobility shift assay
- ntcp
-
STAT5-activated sodium-dependent taurocholate cotransporter gene
- GST
-
glutathione S-transferase
- SH2
-
Src homology 2
-
- Received August 20, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
