Clk/STY (cdc2-like kinase 1) and Akt regulate alternative splicing and adipogenesis in 3T3-L1 pre-adipocytes

• Published in: PloS one 2013-01, Vol.8 (1), p.e53268-e53268
• Main Authors: Li, Pengfei, Carter, Gay, Romero, Jacqueline, Gower, Kathryn M, Watson, James, Patel, Niketa A, Cooper, Denise R
• Format: Article
• Language: English
• Subjects: 3T3-L1 Cells - cytology; 3T3-L1 Cells - metabolism; Adipocytes; Adipocytes - cytology; Adipocytes - metabolism; Adipogenesis; Adiponectin; AKT protein; Alternative Splicing; Animals; Arginine; Binding Sites; Biology; Cdc2 protein; Cells (biology); Deoxyribonucleic acid; DNA; Enzymes; Gene expression; Gene Expression Regulation, Neoplastic; Glucose transport; Growth factors; Health aspects; Hormones; Immunofluorescence; Insulin resistance; Kinases; Medicine; Mice; Microscopy; Mutation; Nuclear Proteins - metabolism; Nuclei; Phosphorylation; Protein kinase C; Protein Kinase C - metabolism; Protein Kinase C beta; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Protein-Tyrosine Kinases - antagonists & inhibitors; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Proteins; Proto-Oncogene Proteins c-akt - metabolism; RNA, Messenger - genetics; RNA, Small Interfering - genetics; RNA-Binding Proteins - metabolism; Serine; Serine-Arginine Splicing Factors; Signaling; siRNA; Splicing factors; Stem cells; Substrates; Thiazoles - pharmacology; Transcription factors; Veterans
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0053268

The development of adipocytes from their progenitor cells requires the action of growth factors signaling to transcription factors to induce the expression of adipogenic proteins leading to the accumulation of lipid droplets, induction of glucose transport, and secretion of adipokines signaling metabolic events throughout the body. Murine 3T3-L1 pre-adipocytes sequentially express all the proteins necessary to become mature adipocytes throughout an 8-10 day process initiated by a cocktail of hormones. We examined the role of Clk/STY or Clk1, a cdc2-like kinase, in adipogenesis since it is known to be regulated by Akt, a pivotal kinase in development. Inhibition of Clk1 by a specific inhibitor, TG003, blocked alternative splicing of PKCβII and expression of PPARγ1 and PPARγ2. SiRNA depletion of Clk1 resulted in early expression of PKCβII and sustained PKCβI expression. Since Clk1 is a preferred Akt substrate, required for phosphorylation of splicing factors, mutation of Clk1 Akt phosphorylation sites was undertaken. Akt sites on Clk1 are in the serine/arginine-rich domain and not the kinase domain. Mutation of single and multiple sites resulted in dysregulation of PKCβII, PKCβI, and PPARγ1&2 expression. Additionally, adipogenesis was blocked as assessed by Oil Red O staining, adiponectin, and Glut1 and 4 expression. Immunofluorescence microscopy revealed that Clk1 triple mutant cDNA, transfected into pre-adipocytes, resulted in excluding SRp40 (SFSR6) from co-localizing to the nucleus with PFS, a perispeckle specific protein. This study demonstrates the role of Akt and Clk1 kinases in the early differentiation of 3T3-L1 cells to adipocytes.