Gernot Walter

Affiliation: UCSD SOM

Professor of Pathology

Biography

Dr. Walter received his Ph.D. in 1965 from the University of Munich/Germany at the Max- Planck-Institute for Biochemistry. He conducted postdoctoral research in Dr. Wolfram Zillig's laboratory at the Max-Planck-Institute for Biochemistry, in Dr. Jake Maizel's laboratory at the Albert Einstein College of Medicine, and in Dr. Renato Dubecco's laboratory at the Salk Institute. In 1972, Dr. Walter joined the faculty of the Salk Institute where he stayed until 1979. From 1979 until 1983 he was professor at the University of Freiburg/Germany, and in 1983 he came to the University of California at San Diego as professor in the department of pathology. Dr. Walter is known for his work on viral tumor antigens and the cellular proteins these antigens interact with in order to transform normal cells into tumor cells. He pioneered the technique of raising antibodies against synthetic peptides, which is widely used to investigate proteins.

Research Summary

Introduction

The study of polyoma viruses has greatly enhanced our knowledge of carcinogenesis. Neoplastic transformation by these viruses results from complex formation between the viral tumor (T) antigens and cellular proteins that are either tumor suppressors or the products of proto-oncogenes. The former become functionally inactivated whereas the latter become activated by complex formation. Other cellular proteins that interact with T antigens are involved in signal transduction, cell cycle control, or apoptosis.

In 1979, our laboratory discovered that antibodies can be generated against proteins by immunization with short synthetic peptides coupled to a protein carrier. We demonstrated that these peptide antibodies can be used to purify proteins by affinity chromatography. From 1985-1987, two cellular proteins of 65-kDa and 36-kDa, which are associated with polyoma virus middle T antigen, were identified by the use of antipeptide antibodies. Importantly, the association between middle T antigen and these proteins is a prerequisit for transformation. In 1989, we cloned and sequenced the cDNA encoding the 65-kDa protein, which has a unique structure consisting of 15 nonidentical repeats. In 1990, David Pallas and collaborators as well as our laboratory, in collaboration with Marc Mumby at the University of Texas at Dallas, demonstrated that the 65-kDa and 37-kDa proteins correspond to the regulatory A and catalytic C subunits, respectively, of protein phosphatase 2A (PP2A). In the last decade, we investigated the structure of the A subunit and how this protein interacts with the catalytic C subunit, regulatory B subunits, and T antigens. We also studied the function of PP2A in vivo. At present, our research focuses on the role of PP2A in tumor formation and cell cycle control.

Subunit Composition and Structure of PP2A

PP2A has three subunits, A, B, and C. The PP2A core enzyme consists of a 36-kDa catalytic C subunit and a 65-kDa regulatory A subunit. One of several B subunits can bind to the core enzyme, and the heterotrimer composed of the A, B, and C subunits is called holoenzyme. The A subunit exists as two isoforms (a and b ), which were first cloned and sequenced in our laboratory. The C subunit also exists as two isoforms (a and b ). The B subunits fall into four families, designated B, B' (also called B56), B'', and B''' which appear unrelated by sequence alignment. The B family has four members, Ba, Bb, Bg, and Bd. The B1 family consists of five genes encoding B'a, B'b, B'g, B'd, B'e. There are a total of at least eight B' subunits including isoforms and splice variants. The B'' family has four members, designated PR48, PR59, PR72 and PR130. The latter two are splice variants of the same gene. The B''' family has two members, striatin and SG2NA. The existence of so many regulatory subunits suggests that PP2A is a highly regulated phosphatase and that its various forms fulfill numerous distinct functions. Another class of proteins able to associate with PP2A core enzyme are tumor (T) antigens encoded by polyoma viruses. Their binding domains on the A subunit overlap with those of B subunits. T antigens play a key role in neoplastic transformation by polyoma viruses, and their association with PP2A is essential for transformation.

To elucidate the function of PP2A, it is important to understand how its subunits interact with each other and what controls the interaction. A model of the core- and holoenzymes, and of complexes of the core enzyme with T antigens is shown below. The A subunit consists of 15 nonidentical repeats. Each repeat is composed of two alpha helices connected by a loop (intra-repeat loop). Adjacent repeats are connected by inter-repeat loops. Collectively, the repeats form an extended, hook-shaped molecule that is stabilized by hydrophobic interactions. The intra-repeat loops are involved in binding B and C subunits as well as T antigens. The B subunits bind to repeats 1-10 and the C subunits to repeats 11- 15 of the A subunit. SV40 small T binds to repeats 3-6 and polyoma virus small T and middle T bind to repeats 2-8.

The question how the different B subunit family members and T antigens, although largely unrelated by sequence, bind to overlapping regions of the A subunit has been approached by site-directed mutagenesis, showing that some Aa mutations in loops 1-10 affect binding of all B subunits (B, B' and B'') whereas others affect binding of specific B subunits. Thus, the binding sites on the A subunit for different types of B subunits are composed of both common and distinct amino acids. Interestingly, a recent study by Virshup et al. revealed that all members of the B, B', and B'' families, but not B''', share two evolutionary conserved domains, 103 and 58 residues in length, which are involved in binding to Aa. The limited homology of these domains escaped detection when the complete sequences of B, B', and B'' were aligned.

Role of PP2A in Human Cancer

Early observations that okadaic acid is both a strong inhibitor of PP2A and a potent tumor promoter indicated that PP2A might be a tumor suppressor, although direct evidence has been lacking. In 1998, Wang and collaborators reported that the gene encoding the Ab subunit of PP2A, located on chromosome 11q23 in a region showing a high frequency of loss of heterozygosity (LOH), was altered in 15% of primary lung tumors, in 6% of lung tumor-derived cell lines, and in 15% of primary colon tumors. Furthermore, Calin and collaborators reported mutations in the genes encoding the Aa and Ab subunits. These studies support the idea that PP2A is a tumor suppressor whose function is destroyed by mutation.

The key question raised by the discovery of mutations in the Aa and Ab genes in human cancer is whether these mutations are relevant for the tumorigenic process. To test the idea that the mutants may be defective in subunit interaction, we constructed them by site- directed mutagenesis and assayed for their ability to associate with different forms of B subunits (B, B', B'') or with the catalytic C subunit. We found that nearly all mutants are defective in binding either B, or B and C subunits, suggesting that certain forms of the PP2A holoenzyme act as tumor suppressors. A remarkable finding was that two Aa subunit mutants, Glu64->Asp in lung carcinoma and Glu64->Gly in breast carcinoma, are defective in B' but normal in B, B'', and C subunit binding. Whether the loss of B' subunit binding might cause loss of tumor suppressor activity, and whether B' itself or a B'- containing holoenzyme is in fact the tumor suppressor are open questions, which we are currently trying to answer.

PP2A is Required for DNA Replication

The initiation of chromosomal DNA replication in eukaryotes can be divided into two general steps. The first occurs during the G1 phase of the cell cycle and involves the formation of a pre-replication complex (pre-RC) containing the origin recognition complex (ORC), Cdc6, Cdt1 and MCM. At the G1/S transition, cyclin-dependent kinase (Cdks) and the Cdc7-Dbf4 kinase convert the pre-RC into an active replication fork by a currently unknown mechanism. There is evidence that the substrate of Cdc7-Dbf4 may be the MCM complex, whereas the Cdk substrates are not known. To investigate whether PP2A is involved in the replication of chromosomal DNA, we used the Xenopus cell-free replication system. We found that removal of PP2A from egg extract caused complete inhibition of DNA replication, and that PP2A is required for initiation but not elongation of DNA replication.

Recently, we demonstrated that immunodepletion of PP2A from egg extracts and inhibition of PP2A activity by okadaic acid abolish loading of initiation factor Cdc45 to the pre-RC. Consistent with a defect in Cdc45 loading, origin unwinding and the loading of RPA and DNA polymerase a are also inhibited. Inhibition of PP2A has no effect on MCM10 loading and on Cdc7-Dbf4 or Cdk2 activity. The substrate of PP2A is neither a component of the pre-RC nor Cdc45. Instead, our data suggest that PP2A functions by dephosphorylating and activating a soluble factor that is required to recruit Cdc45 to the pre-RC. Furthermore, PP2A appears to counteract an unknown inhibitory kinase that phosphorylates and inactivates the same factor. Thus, the initiation of eukaryotic DNA replication is regulated at the level of Cdc45 loading by a combination of stimulatory and inhibitory phosphorylation events. Our model for the role of PP2A is shown below.

References

References From PubMed (NCBI)