BIOLOGICAL OVERVIEW

Before any discussion of Twins function, it will prove useful to have a look at its somewhat complicated enzymatic structure. Twins is the B-subunit (PR55) of a heterotrimeric protein phosphatase known as 2A (PP2A). The trimer is composed of three subunits -- the so-called A and B subunits, plus a catalytic subunit: mammalian PP2A then, is composed of a catalytic 36-kD subunit, a 65kD regulatory subunit (the A subunit or PR65) and the B-subunit. The dimeric core complex (catalytic and A-subunit) may be combined with one of a number of regulatory subunits (the B-subunits) ranging in size from 54 to 74kD. The activity and substrate specificity of the core complex is modulated by association with a B-subunit. In Drosophila, genes for the catalytic and PR65 (A) subunits of PP2A were cloned by sequence similarity to their mammalian counterparts (Mayer-Jaekel, 1992). Cloning of the B subunit PR55, coding for Twins protein, was accomplished through homology to the human homolog (Mayer-Jaekel, 1993 and Uemura, 1993).

Twins protein and the catalytic subunit of PP2A, the two proteins for which mutations have been isolated, have a multitude of functions, suggesting involvement in a number of pathways. Initially implicated as a regulator of mitotic progression (see Effects of mutation and Evolutionary homologs sections) , Twins has subsequently been implicated in three additional roles.

In the first of these, twins mutation causes a peripheral nervous system defect similar to that observed in numb and musashi mutations. How does this defect manifest itself? At this point, a quick look at the non-mutated phenotype is in order. Adult mechanosensory bristles normally consist of four cells: two cells of neural derivation (neuron and glial or thecogen cells), and two support cells ( a socket cell [tormogen] and a shaft cell [trichogen]). These four cells are derived from a single precursor called the sensory organ precursor (SOP). Mutations in twins (Shiomi, 1994), musashi (Nakamura, 1994) and numb result in an increase in the ratio of support cells, at the expense of neural cells.

One additional digression will clarify Twins's involvement in such defects, with respect to numb, taking us by way of tramtrack: mutation of tramtrack results in the the transformation of socket and shaft cells to neuron and glial cells. Ectopic expression of ttk has just the opposite effect: the transformation of neuron and glial cells into socket and shaft cells. Numb protein is asymmetrically distributed to neural precursor cells in the first division of the SOP, and Numb targets tramtrack, which then acts as a repressor of support cell fate in neural and glial progeny (Rhyu, 1994 and Guo, 1995). Interestingly, an N-terminal domain of Numb protein consists of residues predictive of a phosphotyrosine binding domain (PTB domain) (Uemura, 1989 and Zhong, 1996). It is possible that Twins protein regulates a Numb phosphoprotein target, and in this manner is involved in the peripheral nervous system defect.

A second function for Twins is found in wing morphogenesis. Mutation of twins causes a pattern duplication in Drosophila imaginal discs. Inactivation of twins induces the formation of extra wing blade anlagen in the posterior compartment. The duplication is mirror symmetrical, and the line of symmetry does not correspond to any of the known compartment borders (Uemura, 1993).

A third function for Twins has to do with eye morphogenesis. Mutations in the catalytic subunit of PP2P interfere with Ras pathway function in eye morphogenesis. PP2P mutation exacerbates defects caused by unregulated Ras function and diminishes defects cause by unregulated Raf function. Ras and Raf are both components of the EGF-receptor signaling pathway required for determination of photoreceptor fate in the compound eye (Wassarman, 1996).

These four defects, associated with mutant PP2A (defective mitosis, defective cell fate determination in the PNS, alteration of patterning in the wing, and interferance in Ras pathway function in eye morphogenesis) do not seem to share any common denominator, other than the involvement of PP2A. It is likely that PP2A substrates are different for each of these defects.

One of the most exciting aspects of PP2A research is PP2A involvement in differentiation. In terms of understanding developmental roles, Drosophila is the organism of choice, but in terms of an understanding of biochemical pathways, a multitude of other organisms are proving useful. Organisms as diverse as yeast, Acetabularia, Xenopus and mammals reveal an intimate interaction between PP2A in the cytoskeleton and cyclinB/cdc2. Different PP2A subunits in mammals react differently with respect to subcellular location, suggesting cell type specific functions (McCright, 1996). Other work shows that PP2A is involved in positively and negatively regulating response to neuropeptides with different responses depending on receptor subtype (Huang, 1996). Finally, PP2A plays a key role in the differentiation-dependent expression of the neurofilament gene, suggesting an involvement in neural cell fate (Sasahara, 1996). It is clear that PP2A takes on many developmental roles, and only the combination of investigative genetics and the resources of developmental biology coupled with biochemical techniques will reveal the complexities of PP2A's functions.

GENE STRUCTURE

Exons - 7

PROTEIN STRUCTURE

Amino Acids - 499 for DPR55-1 and 443 for DPR55-4

Structural Domains

The cDNAs for Twins proteins fall into two classes: the DPR55-1-type and the DPR55-4-type. Three exons (V, VI and VII) are common to all of the cDNAs. The 5' sequences of the DPR55-1-type and the the DPR55-4-type are derived from (respectively) exons I and II and III and IV. There are additional variations in this latter class, in which varying portions of the 3' end of exon III are present. The DPR55-4 protein differs only in the first 3 amino acids from the DPR55-1 protein sequence and shows 78% and 77% identity to the human PR55alpha and PR55beta sequences, respectively. The sequence alignment with the S. cerevisiae PR55 homolog CDC65 requires the introduction of several gaps but still shows 72% similarity if conserved amino acid substitutions are considered (Mayer-Jaekel, 1993).

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit ofprotein phosphatase 2A (PR65) from Drosophila have been isolated byhomology screening with the corresponding human cDNAs. The Drosophila cloneswere used to analyze the spatial and temporal expression of the transcripts encodingthese two protein subunits. The Drosophila PR65 cDNA clones contains an open readingframe of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acidsequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms,respectively. As previously reported for the mammalian PR65 isoforms, DrosophilaPR65 is composed of 15 imperfect repeating units of approximately 39 amino acids.The residues contributing to this repeat structure also show the highest sequenceconservation between species, indicating a functional importance for these repeats.The gene encoding Drosophila PR65 was located at 29B1,2 on the secondchromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit andtwo transcripts of 1.6 and 2.5 kb encoding the catalytic subunit can be detectedthroughout Drosophila development. All of these mRNAs are most abundant duringearly embryogenesis and are expressed at lower levels in larvae and adult flies. Insitu hybridization of different developmental stages showed a colocalization of thePR65 and catalytic subunit transcripts. The mRNA expression is high in the nursecells and oocytes, consistent with a high equally distributed expression in earlyembryos. In later embryonic development, the expression remains high in the nervoussystem and the gonads but the overall transcript levels decrease. In third instar larvae,high levels of mRNA can be observed in the brain, imaginal discs, and salivary glands.These results indicate that protein phosphatase 2A transcript levels change duringdevelopment in both a tissue and in a time-specific manner (Mayer-Jaekel, 1992).

date revised: 15 FEB 97 

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