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Inactivation of smad-transforming growth factor beta signaling by Ca(2+)-calmodulin-dependent protein kinase II
S J Wicks1, S Lui, N Abdel-Wahab
1Department of Cancer Medicine, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London W12 ONN, United Kingdom.
Abstract:
Members of the transforming growth factor beta (TGF-beta) family transduce signals through Smad proteins. Smad signaling can be regulated by the Ras/Erk/mitogen-activated protein pathway in response to receptor tyrosine kinase activation and the gamma interferon pathway and also by the functional interaction of Smad2 with Ca(2+)-calmodulin. Here we report that Smad-TGF-beta-dependent transcriptional responses are prevented by expression of a constitutively activated Ca(2+)-calmodulin-dependent protein kinase II (Cam kinase II). Smad2 is a target substrate for Cam kinase II in vitro at serine-110, -240, and -260. Cam kinase II induces in vivo phosphorylation of Smad2 and Smad4 and, to a lesser extent, Smad3. A phosphopeptide antiserum raised against Smad2 phosphoserine-240 reacted with Smad2 in vivo when coexpressed with Cam kinase II and by activation of the platelet-derived growth factor receptor, the epidermal growth factor receptor, HER2 (c-erbB2), and the TGF-beta receptor. Furthermore, Cam kinase II blocked nuclear accumulation of a Smad2 and induced Smad2-Smad4 hetero-oligomerization independently of TGF-beta receptor activation, while preventing TGF-beta-dependent Smad2-Smad3 interactions. These findings provide a novel cross-talk mechanism by which Ca(2+)-dependent kinases activated downstream of multiple growth factor receptors antagonize cell responses to TGF-beta.
Insights
Calcium-dependent protein kinase II (Cam kinase II) antagonizes transforming growth factor beta (TGF-beta) signaling by preventing Smad protein nuclear accumulation and interactions. This cross-talk mechanism, activated by various growth factors, impacts cellular responses to TGF-beta.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Signal transduction
Background:
- Transforming growth factor beta (TGF-beta) signaling is crucial for cellular processes and mediated by Smad proteins.
- Smad signaling is regulated by pathways including Ras/Erk and gamma interferon, and Smad2 interacts with Ca(2+)-calmodulin.
- Cross-talk between different signaling pathways allows for complex cellular regulation.
Purpose of the Study:
- To investigate the inhibitory effect of Ca(2+)-calmodulin-dependent protein kinase II (Cam kinase II) on TGF-beta/Smad signaling.
- To identify Smad proteins as direct or indirect targets of Cam kinase II.
- To elucidate the mechanism by which Cam kinase II antagonizes TGF-beta responses.
Main Methods:
- In vitro kinase assays to determine Smad2 phosphorylation sites by Cam kinase II.
- In vivo studies involving co-expression of Cam kinase II with Smad proteins.
- Phosphopeptide antiserum generation and Western blot analysis to detect phosphorylated Smad2.
- Confocal microscopy to assess Smad2 nuclear localization and Smad complex formation.
Main Results:
- Constitutively active Cam kinase II expression prevents TGF-beta-dependent transcriptional responses.
- Cam kinase II directly phosphorylates Smad2 at specific serine residues (S110, S240, S260) in vitro.
- Cam kinase II induces in vivo phosphorylation of Smad2 and Smad4, and to a lesser extent Smad3.
- Cam kinase II inhibits Smad2 nuclear accumulation and Smad2-Smad4 hetero-oligomerization independently of TGF-beta receptor activation.
- Cam kinase II prevents TGF-beta-induced Smad2-Smad3 interactions.
Conclusions:
- Ca(2+)-calmodulin-dependent protein kinase II acts as a negative regulator of TGF-beta/Smad signaling.
- Cam kinase II antagonizes TGF-beta responses by interfering with Smad protein complex formation and nuclear translocation.
- This represents a novel cross-talk mechanism where Ca(2+)-dependent kinases activated by diverse growth factor receptors can inhibit TGF-beta cellular effects.