|Ph.D Student||Barr Haim|
|Subject||A Study of Protein Interactions Involving the B55 Subunit|
of Protein Phosphatase 2A
|Department||Department of Medicine||Supervisor||Professor Tamar Kleinberger|
Protein Phosphatase 2A (PP2A) is a heterotrimeric enzyme found in all eukaryotic cells. The holoenzyme is composed of a catalytic subunit (C) and two regulatory subunits (A, B subunits). The A subunit is primarily of structural importance, however the B subunit has been considered to be the primary targeting component of PP2A.This is supported by the existence of multiple and unrelated B subunit families, and the fact that several viral antigens interact with, or displace, B subunits from the PP2A holoenzyme during viral infection or transformation. Adenovirus protein E4orf4 interacts specifically with the B55 subunit, and requires PP2A for induction of its known activities, primarily apoptosis. In the hope that we will shed light on the function of PP2A in the cell, we have attempted to isolate cellular proteins participating in complexes formed specifically by B55. We have used the Ras Recruitment System (RRS) which is an alternative to the well-known yeast two-hybrid system, to genetically screen a HeLa cDNA library for proteins interacting with PP2A B55 subunit. Using B55-RRS we isolated seven distinct cDNAs encoding mammalian proteins never-before described as B55-interacting proteins. The RING finger protein-interaction motif was present in two of the seven proteins, HARI-2, and DinG. A third protein called UIP28, which is related to HARI-2 was also identified as interacting with B55. RING finger proteins are prominent in the ubiquitin system of regulated protein degradation. The RING motif of E3 ubiquitin ligases has been extensively studied as the region responsible for mediating the interaction between ubiquitin conjugating enzymes (E2s) and the E3, which acts as a scaffold to juxtapose the target protein with the E2. We have found that B55-3xHA was rapidly degraded in a proteasome-dependent process. We have explored the possibility that B55 is a target for ubiquitylation in mammalian cells, and the roles of B55-interacting RING finger proteins in this process. However, overexpression of the murine homolog of HARI-2 and UIP28 did not affect B55 ubiquitylation.
PP2A has been found to interact with several protein kinases through the action of scaffold proteins so that a kinase/phosphatase module is formed. We have found that PP2A interacts with PKCz through a mutual interaction with UIP28, acting as a scaffold. Formation of the PP2A/PKCz complex by overexpression of UIP28 and B55 in NIH 3T3 cells reverses the activation of an NF-kB-luciferase reporter gene which has been shown to be activated by PKCz. Overexpression of B55 or UIP28 alone is not sufficient to decrease luciferase activity.
PKCz is also a substrate for ubiquitylation. We have shown that overexpression of PP2A or UIP28 prevents ubiquitylation of PKCz in proteasome-inhibited HEK cells. This process appears to be functionally distinct from the down-regulation of
NF-kB-reporter activity by UIP28 with B55.