SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms.
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)).
Ram
P A
PA
Department of Biology, Division of Cell Biology, Boston University, Boston, Massachusetts 02215, USA.
Waxman
D J
DJ
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007016:Hypophysectomy; D017874:Immediate-Early Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008894:Milk Proteins; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D011986:Receptors, Somatotropin / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D012097:Repressor Proteins; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014162:Transfection; D018909:src Homology Domains
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D013006:Growth Hormone; D017874:Immediate-Early Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008894:Milk Proteins; D011506:Proteins; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D050799:STAT5 Transcription Factor; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C108944:SOCS1 protein, human; C106643:SOCS2 protein, human; C106644:SOCS3 protein, human; C494095:STAT5B protein, human; C491589:Socs1 protein, rat; C491408:Socs2 protein, rat; C491410:Socs3 protein, rat; C494097:Stat5b protein, rat