Xiaoqing Sun

Functional characterization of human PFTK1 as a cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle whose activities are controlled by associated cyclins. PFTK1 shares limited homology to CDKs, but its ability to associate with any cyclins and its biological functions remain largely unknown. Here, we report the functional characterization of human PFTK1 as a CDK. PFTK1 specifically interacted with cyclin D3 (CCND3) and formed a ternary complex with the cell cycle inhibitor p21Cip1 in mammalian cells. We demonstrated that the kinase activity of PFTK1 depended on CCND3 and was negatively regulated by p21Cip1. Moreover, we identified the tumor suppressor Rb as a potential downstream substrate for the PFTK1/CCND3 complex. Importantly, knocking down PFTK1 expression by using siRNA caused cell cycle arrest at G1, whereas ectopic expression of PFTK1 promoted cell proliferation. Taken together, our data strongly suggest that PFTK1 acts as a CDK that regulates cell cycle progression and cell proliferation.

The X-linked lymphoproliferative disease gene product SAP associates with PAK-interacting exchange factor and participates in T cell activation

SLAM (signaling lymphocyte activation molecule)-associated protein (SAP) is a Src homology 2 (SH2) domain-containing adaptor expressed in T cells and natural killer cells. Its essential role in immune responses is underscored by the recent finding that mutations in SAP result in a rare but fatal X-linked lymphoproliferative disease (XLP). Although SAP is known to associate with SLAM-family receptors, the exact molecular mechanism by which SAP regulates lymphocyte signaling remains elusive. We here report that in T cells, SAP associates with the PAK-interacting exchange factor (PIX), a guanine nucleotide exchange factor (GEF) specific for Rac/Cdc42 GTPases. Moreover, SAP, PIX, and an activated form of Cdc42 form a complex in mammalian cells. We demonstrate that the SAP-PIX interaction is specific and is mediated by the C-terminal region of the SAP SH2 domain and the PIX SH3 domain. We further show that SAP is required for the recruitment of PIX to the SLAM-family receptors. Interestingly, overexpression of SAP, but not its homolog EAT-2, leads to a synergistic activation of nuclear factor of activating T cells (NFAT) in combination with a calcium signal in T cells. This SAP-mediated activation appears to be receptor-dependent and can be blocked by a dominant negative form of PIX. Taken together, our data strongly suggest that, in addition to the known SAP-interacting kinase Fyn, PIX may be another key player in SAP-mediated T cell activation.

Proteomic analysis of ubiquitinated proteins in normal hepatocyte cell line Chang liver cells

Post‐translational modification by ubiquitin (Ub) and Ub‐like modifiers is one of the most important mechanisms regulating a wide range of cellular processes in eukaryotes. Through mediating 26S proteasome‐dependent degradation of substrates, the covalent modification of proteins by multiple Ub (ubiquitination) can regulate many different cellular functions such as transcription, antigen processing, signal transduction and cell cycle. To better understand ubiquitination and its functions, proteomic approaches have been developed to purify and identify more protein substrates. The S5a subunit of the 26S proteasome binds to poly‐Ub chains containing four or more Ub. In this study, immobilized GST‐S5a fusion protein was used to affinity‐purify ubiquitinated proteins from Chang liver cells. The purified proteins were then identified with multi‐dimensional LC combined with MS/MS. Eighty‐three potential ubiquitination substrates were identified. From these proteins, 19 potential ubiquitination sites on 17 potential substrates were determined. These potential ubiquitination substrates are mainly related to important cellular functions including metabolism, translation and transcription. Our results provide helpful information for further understanding of the relationship between ubiquitination machinery and different cell functions.

Adapter protein NRBP associates with Jab1 and negatively regulates AP-1 activity

Jun activation domain‐binding protein 1 (Jab1) is a coactivator of activating protein‐1 (AP‐1) and is the fifth component of the COP9 signalosome complex. It interacts with a variety of proteins and plays important roles in diverse signaling pathways and cellular function including oncogenesis. We show here that Jab1 interacts in vivo with nuclear receptor binding protein (NRBP), an evolutionarily conserved adapter protein with a kinase‐like domain. We further show that NRBP inhibits Jab1‐induced phosphorylation of c‐Jun and AP‐1 activation. Finally, overexpression of NRBP in mammalian cells specifically inhibits AP‐1 activation by various stimuli. Taken together, our data suggest that NRBP may be an important negative regulator of Jab1‐mediated functions such as gene transcription and tumor progression.