BIOLOGICAL OVERVIEW

The metazoan Wee1-like kinases Wee1 and Myt1 regulate the essential mitotic regulator Cdk1 by inhibitory phosphorylation. This regulatory mechanism, which prevents Cdk1 from triggering premature mitotic events, is also induced during the DNA damage response and used to coordinate cell proliferation with crucial developmental events. Despite the role for Myt1 regulation of Cdk1 during meiosis, relatively little is known of how Myt1 functions at other developmental stages. To address this issue, a functional analysis was undertaken of Drosophila Myt1 that has revealed novel developmental roles for this conserved cell cycle regulator during gametogenesis. Notably, more proliferating cells were observed in myt1 mutant testes and ovaries than controls. This can partly be attributed to ectopic division of germline-associated somatic cells in myt1 mutants, suggesting that Myt1 serves a role in regulating exit from the cell cycle. Moreover, mitotic index measurements suggest that germline stem cells proliferate more rapidly in myt1 mutant females. In addition, male myt1 germline cells occasionally undergo an extra mitotic division, resulting in meiotic cysts with twice the normal numbers of cells. Based on these observations, it is proposed that Myt1 serves unique Cdk1 regulatory functions required for efficient coupling of cell differentiation with cell cycle progression. Myt1 shares two conserved domains with other Myt1 kinases, that are not present in nuclear Wee1 kinases (Lamitina, 2002; Liu, 1997; Wells, 1999), a potential trans-membrane domain and a C-terminal putative Cyclin B interaction motif. The results are consistent with the idea that Myt1 specifically regulates Cdk1 activity in the cytoplasm, in contrast to Wee1, whose functions are nuclear (Jin, 2005).

A conserved molecular mechanism that controls entry into mitosis in eukaryotic cells involves the activation of a cyclin-dependent kinase called Cdk1, which serves as a master regulator of early mitotic events. To exit mitosis, Cdk1 activity must then be eliminated by proteolytic degradation of its cyclin subunit. Once mitotic cyclins are re-synthesized during interphase, Cdk1 is maintained in an inactivate state by inhibitory phosphorylation, preventing premature initiation of mitotic events during S phase or when the DNA damage checkpoint is induced, to allow time for DNA repair. This Cdk1 regulatory mechanism is also used to coordinate the timing of mitosis with morphogenetic cell movements, as was first demonstrated by studies of the intricate spatial and temporal pattern of cell division during Drosophila gastrulation. Ectopic expression and morpholino depletion experiments carried out in Xenopus also suggest that Cdk1 regulatory mechanisms involving Wee1 kinases couple cell division with cell movements during gastrulation, implying that this is a conserved developmental mechanism (Leise, 2002; Murakami, 2004; Jin, 2005 and references therein).

Two related classes of Cdk1 inhibitory kinases have been identified in metazoans: Wee1 and Myt1. Xenopus and C. elegans each contain two Wee1-like kinases and a single Myt1 ortholog, each of which exhibits distinct expression patterns during development (Lamitina, 2002; Leise, 2002; Murakami, 2004; Nakanishi, 2000; Okamoto, 2002; Wilson, 1999). The situation is somewhat simpler in Drosophila, which has a single Cdk1 inhibitory kinase of each type: Wee1 and Myt1. Drosophila Wee1 is a nuclear kinase that is essential for regulating Cdk1 during the rapid, maternally controlled S/M nuclear divisions of early embryogenesis; however, Wee1 is otherwise dispensable for zygotic development. Myt1 localizes to Golgi and endoplasmic reticulum membranes, as also reported for the Xenopus and human Myt1 orthologs (Booher, 1997; Liu, 1997; Mueller, 1995). The Myt1 kinases were originally characterized as membrane-associated dual-specificity Cdk1 kinases that phosphorylate a threonine (T14) residue of Cdk1, as well as the Y15 site that is also targetted by nuclear Wee1 kinases (Booher, 1997; Liu, 1997; Mueller, 1995). These differences in Wee1 and Myt1 protein localization and target site specificity suggest that the metazoan Cdk1 inhibitory kinases have evolved distinct cell cycle regulatory functions required at different stages of development (Jin, 2005).

Transgenic overexpression and RNAi experiments involving Myt1 suggest that its expression primarily affects the G2 phase of the cell cycle; however, genetic evidence of specific functions for Drosophila Myt1 that are essential for normal development has been lacking (Cornwell, 2002; Price, 2002). Previously, biochemical studies of the prolonged 'G2-like' growth state of immature oocytes suggest that Myt1 is responsible for inhibitory phosphorylation of Cdk1 during this stage of female meiosis in Xenopus (Furuno, 2003; Karaiskou, 2004; Nakajo, 2000; Palmer, 1998; Peter, 2002) and in the starfish A. pectinifera (Okano-Uchida, 2003; Okumura, 2002). Mutations affecting a C. elegans homolog have also implicated Myt1 in the regulation of male meiosis (Lamitina, 2002). Mutations affecting Drosophila Myt1 have been isolated and characterized. These studies reveal that Myt1 serves regulatory functions that have not previously been described but are important for both mitotic and meiotic cell cycles during gametogenesis. These observations implicate Myt1 in specific Cdk1 regulatory mechanisms that are required for coordinating cell cycle behavior with crucial developmental transitions (Jin, 2005).

Regulation of Cdk1 by inhibitory phosphorylation is critical for cell survival and for the developmental regulation of cell proliferation. Drosophila Wee1 serves an essential role in a pre-mitotic checkpoint that operates during the rapid S/M nuclear divisions of early embryogenesis. The cell cycle timing defects observed in these mutants are consistent with the proposal that Wee1 serves a conserved role in protecting nuclei from cytoplasmically activated Cdk1. Myt1 serves distinct cell cycle regulatory functions, required for the fidelity of specific stages of development (Jin, 2005).

Overexpression studies in several systems suggest that Myt1 can influence the timing of G2/M transitions (Cornwell, 2002; Price, 2002; Wells, 1999). Loss-of-function studies now demonstrate that Myt1 serves distinct Cdk1 regulatory functions that are essential for normal gametogenesis and for adult bristle development. Consistent with previous studies in other organisms that implicated Myt1 in female meiosis (Kalous, 2005; Karaiskou, 2004; Okano-Uchida, 2003; Okumura, 2002; Palmer, 1998; Peter, 2002), a marked elevation of meiotic chromosome segregation defects is observed in the progeny of female myt1 mutants. Female myt1 mutants are fertile in spite of these segregation defects; however, many of their progeny undergo variable embryonic lethality. In male myt1 mutants, loss of Myt1 function results in complete sterility. This phenotype appears to be due to defects during both meiosis and spermatid differentiation in male myt1 mutants. Normally, Drosophila spermatocytes undergo a prolonged G2 phase arrest, which allows time to synthesize cellular components required for subsequent development, before the onset of meiosis I cell division. Oocytes do not undergo a similar growth phase, owing to specialization of the germline nurse cells, which synthesize the mRNAs and proteins required for egg development. Thus, unlike spermatocytes, Drosophila oocytes almost immediately progress into prophase of meiosis I after completing the four mitotic divisions that produce the 16-cell cyst. These differences in male and female germline development may explain the differences in requirements for Myt1 activity observed. Moreover, the data suggest that Myt1 has evolved specific functions that are important for developmentally regulated growth phases. This hypothesis is also consistent with the requirement for Myt1 in Xenopus and A. pectinifera during the prolonged 'G2-like' prophase arrest of early oocytes, which also involves extensive cell growth and synthesis of proteins and mRNAs required for subsequent embryonic development (Furuno, 2003; Karaiskou, 2004; Nakajo, 2000; Okano-Uchida, 2003; Okumura, 2002; Palmer, 1998; Peter, 2002; Jin, 2005 and references therein).

In addition to confirming that Myt1 serves a conserved role in regulating meiosis, these studies of myt1 mutants also provide evidence that Myt1 serves novel functions that affect mitotic cell proliferation during both male and female gametogenesis. Mitotic index measurements of female germline cells suggest that loss of Myt1 activity influences the timing of the mitotic cell cycles that precede meiosis, although live analysis will be needed to verify this conclusion. Delays in mitosis may also contribute to this phenotype; the observed increase in numbers of germline cysts in myt1 mutant germaria was not directly proportional to the increased mitotic index seen in germline stem cells (Jin, 2005).

A further unexpected mitotic defect associated with loss of Myt1 activity involved ectopic divisions of germline-associated somatic cells, seen in both males and females. Because germline-associated somatic cells normally become quiescent as they differentiate, this observation suggests a role for Myt1 in a molecular mechanism that allows or facilitates exit from the cell cycle. An additional mitotic defect was identified in male myt1 mutants, which was not seen in females. Approximately 10% of the cysts undergo an extra round of mitotic cell division before cells differentiate into primary spermatocytes, suggesting that Myt1 also affects the fidelity or timing of this developmentally regulated cell fate decision. However, unlike previously described male-sterile over-proliferation mutants with cell fate defects, the majority of the myt1 mutant cysts do not undergo such ectopic mitotic divisions. Collectively, these data suggest that Myt1 serves distinct Cdk1 regulatory functions that coordinate cell cycle behavior with important developmental transitions. Precisely how Myt1 accomplishes these diverse functions is unknown; however, the results are consistent with the idea that Myt1 specifically regulates Cdk1 activity in the cytoplasm (Jin, 2005).

Strikingly, RNAi depletion of Myt1 in cultured Drosophila cells was previously reported to cause a marked disruption of the Golgi apparatus (Cornwell, 2002). This observation suggests an intriguing possibility. Since the Golgi apparatus serves a key role in trafficking and secretion of proteins in rapidly growing cells, perhaps Myt1 regulation of Cdk1 activity might indirectly affect the biosynthesis and assembly of subcellular structures required for crucial developmental transitions affected in myt1 mutants (Jin, 2005).

According to current models describing regulation of the G2/M transition, Myt1 and Wee1 inhibit Cdk1 in the cytoplasmic and nuclear compartments during interphase, respectively, ensuring a complete block to mitotic progression. Once cells are ready to divide the regulatory proteins that trigger mitosis are thought to first activate Cdk1 at the centrosomes, initiating a self-amplifying wave of Cdk1 activation that sweeps through the cytoplasm and into the nucleus (Jackman, 2003; Kramer, 2004). This mechanism ensures that early mitotic events are coordinated throughout the cell. Loss of Myt1 activity would be expected to disrupt this coordination by allowing premature activation of Cdk1 in the cytoplasmic compartment, even if Wee1 can still protect the nucleus from active Cdk1. Key assumptions of this model can now be tested in vivo, using wee1 and myt1 mutants to determine how loss of these Cdk1 inhibitory kinases affects specific cell structures and organelles, at different stages of the cell cycle (Jin, 2005).

GENE STRUCTURE

cDNA clone length - 1905

Bases in 5' UTR - 524

Exons - 4

Bases in 3' UTR - 307

PROTEIN STRUCTURE

Amino Acids - 533

Structural Domains

The dMyt1 kinase gene encodes a 533-amino acid protein with a predicted molecular weight of 61.2 kDa.The overall sequence identity of dMyt1 kinase to human and Xenopus Myt1 kinases is 33% and 31%, respectively, although, from comparison of the kinase domains, dMyt1 shares 48% identity with both human and Xenopus Myt1. In addition, analysis of hydrophobic regions has identified a putative membrane anchor region that correlates well with the membrane anchor regions of human and Xenopus Myt1 (Cornwell, 2002).

Drosophila myt1 is predicted to encode a 61 kDa protein (533 amino acids). Within the kinase domain, Myt1 shares 49% and 47% amino acid sequence identity with X. laevis and H. sapiens Myt1 homologs, and 31% sequence identity with Wee1 in the same region. Myt1 also shares two conserved domains with other Myt1 kinases, that are not present in nuclear Wee1 kinases: a potential trans-membrane domain and a C-terminal putative Cyclin B interaction motif (Jin, 2005).

date revised: 5 February 2006

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