SOCS/CIS Protein Inhibition of Growth Hormone-stimulated STAT5 Signaling by Multiple Mechanisms

• Published in: The Journal of biological chemistry Vol. 274; no. 50; pp. 35553 - 35561
• Main Authors: Ram, P A, Waxman, D J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.12.1999
• Subjects: Animals; Carrier Proteins - metabolism; Cell Line; Cloning, Molecular; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Growth Hormone - pharmacology; Humans; Hypophysectomy; Immediate-Early Proteins - metabolism; Intracellular Signaling Peptides and Proteins; JAK2 protein; Male; Milk Proteins; Proteins - metabolism; Rats; Rats, Inbred F344; Receptors, Somatotropin - drug effects; Receptors, Somatotropin - physiology; Recombinant Fusion Proteins - drug effects; Recombinant Fusion Proteins - metabolism; Repressor Proteins; Signal Transduction - drug effects; Signal Transduction - physiology; SOCS-1 protein; SOCS-3 protein; SOCS-6 protein; SOCS/CIS protein; src Homology Domains; STAT5 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Trans-Activators - genetics; Trans-Activators - metabolism; Transcription Factors; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.50.35553

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 vitro binding assays were carried out using glutathione S -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)).