Structural insights into the function of the catalytically active human Taspase1

19 pags., 7 figs., 2 tabs.Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics.This project has been funded in whole with Federal funds from the National Cancer Institute (NCI), National Institutes of Health (NIH), under Chemical Biology Consortium contract no. HHSN261200800001E

Regulation of growth hormone signaling by SIRP(alpha) and the protein tyrosine phosphatase SHP-2.

Reversible tyrosyl phosphorylation of proteins is a key regulatory mechanism that cells utilize to transduce signals in response to a variety of polypeptide hormones and growth factors, which regulate cellular growth, movement, or metabolism. Growth hormone (GH) is a key regulator of body growth and metabolism, and GH exerts its physiological actions by binding to GH receptor (GHR) and rapidly and transiently inducing tyrosyl phosphorylation and activation of the Janus tyrosine kinase JAK2. Activation of JAK2 is required for tyrosyl phosphorylation and/or activation of signaling molecules such as GHR, JAK2, the signal transducer and activator of transcription STAT5B and extracellular signal regulated kinases ERKs 1 and 2. Although the events which initiate GH signaling (such as JAK2 activation and tyrosyl phosphorylation) are beginning to be understood, the events which terminate GH signaling (such as dephosphorylation of signaling proteins) are poorly understood. In this dissertation, the protein tyrosine phosphatase SHP-2 and its substrate SIRP a (signal regulatory protein a ) are identified as negative regulators of GH signaling. GH induces tyrosyl phosphorylation of SHP-2 and SIRP a and association of SHP-2 with both SIRP a and GHR. Although JAK2 can associate with and tyrosyl phosphorylate SIRP a in a COS cell overexpression system, neither LiF nor IFN g , ligands that activate JAK2, induces tyrosyl phosphorylation of SIRP a or association of SHP-2 with SIRP a . Overexpression of wild-type SIRP a , but not SIRP a which lacks all potential SHP-2 binding sites, reduces tyrosyl phosphorylation of STAT5B, ERK, and a subset of JAK2 associated with SIRP a . To explore further the role of SHP-2 in GH signaling, the role of the association of SHP-2 with GHR was examined. SHP-2 associates with tyrosine 595 of GHR via the SH2 domains of SHP2, and mutation of tyrosine 595 prolongs GH-promoted tyrosyl phosphorylation of STAT5B and GHR, but not JAK2. These data indicate that SHP-2 and SIRP a may negatively regulate GH signal transduction.Ph.D.Biological SciencesMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131798/2/9929962.pd

Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions

Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK–PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK–PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells