The DYRK Family of Kinases The human genome encodes approximately 518 different kinases in nine distinct families

(Manning et al., 2002). These kinases regulate all aspects of human physiology, including all the

hallmarks of cancer (Hanahan and Weinberg, 2011). Indeed, approximately half of all human kinases

have been implicated in human diseases (Manning et al., 2002). Although some of these kinases (such

as protein kinase A) are among the most intensely studied human proteins, others haven’t been

thoroughly investigated.

The DYRKs are one family of kinases that are found in most eukaryotes, including plants,

animals, fungi and protists. Most kinases can phosphorylate their substrates on either a tyrosine or a

serine/threonine amino acid. This family is a bit unusual as DYRK kinases are “dual specificity” and

can phosphorylate their substrates on any one of a tyrosine, serine or threonine. Among the members

of this family that have been studied, they can phosphorylate themselves on a tyrosine residue, which

regulates the kinase’s activity. Humans contain five DYRK proteins: DYRK1A, DYRK1B, DYRK2,

DYRK3, and DYRK4 (Aranda et al., 2011). The DYRK1A kinase was the first of these proteins to be

cloned and is the most extensively characterized. The gene is located on chromosome 21 seems to

make a key contribution to Down syndrome pathology (Becker and Sippl, 2011). It is a key regulator

of neurological development in animals (Tejedor and Hämmerle, 2011) and plays in role in

neurodegenerative diseases like Alzheimer disease and Parkinson disease (Wegiel et al., 2011). The

DYRK1B kinase helps to keep some cell types quiescent and can inhibit apoptosis, thus suggesting

that it may act as a tumor suppressor (Friedman, 2007). DYRK1A can phosphorylate Caspase-9 and

influence apoptosis in human cells (Seifert et al., 2008). DYRK1A may also be involved in some

human cancers, as it is known to affect the interactions of Ras, BRAF and MEK (Kelly and Rahmani,

2005). Recently, it was demonstrated that DYRK1A is phosphorylated by the LATS2 kinase which in

turn affects DYRK1A’s ability to phosphorylate LIN52 that can associate with pRb and other proteins

(Tschöp et al., 2011). The loss of DYRK1A function diminished the frequency of cells exiting the cell

cycle (Litovchick et al., 2011), consistent with the idea that DYRK1A may function as a human tumor

suppressor protein. In some brain cancers, it appears that inhibition of DYRK1A may help to kill the

cancer cells (Pozo et al., 2013). Thus, depending upon a variety of factors, DYRK1A could either

promote or inhibit cancer development in humans (Birger and Izraeli, 2012). Curiously, DYRK1A

may also be a gene that was critical in the evolution of humans. Comparing the sequence of the human

genome with the sequence of the Neandertal genome shows evidence that alleles of DYRK1A have

been positively selected for as humans evolved (Green et al., 2010). Considering that there were 25

papers published on DYRK1A in the first six months of 2013, this is a very “hot gene” at the moment.

Yak1p in Yeast The budding yeast Saccharomyces cerevisiae has a single member of the DYRK kinase family.

Overall, 22.4% of the amino acids in the yeast Yak1 protein are identical to the human DYRK1A

protein. Within the kinase domain, 38.2% of the amino acids are identical, including key residues

known to be essential for ATP binding and catalysis. The YAK1 gene was the first member of the

DYRK kinases to have been identified, almost 25 years ago. Initially it was cloned as a gene important

for cells to sense and respond to the nutritional environment and named yet another kinase (Garrett and

Broach, 1989). Active Yak1p seems to diminish the growth rate of yeast cells when food is scarce

(Hartley et al., 1994) or when it is overexpressed (Pratt et al., 2007), which has striking parallels to the

more recently identified function of human DYRK1A to decrease cell cycling (Litovchick et al.,

2011).

We know of several different ways in which information gets sent to Yak1p. Both the TOR

pathway (Martin et al., 2004) and the Ras/Protein Kinase A (PKA, Garrett and Broach, 1989) signal

transduction pathways can influence subcellular localization or the activity of Yak1p. These are two of

the major signal transduction pathways that allow a cell to sense and respond to its environment

(Zaman et al., 2008; Broach, 2012), so it makes some sense that they can affect Yak1p. Active Yak1p

can pass its signal on to many other molecules. Recently, it was shown that YAK1 mRNA levels can be

controlled by Whi3p, an RNA-binding protein that helps orchestrate cellular development and

maintain appropriate ploidy levels (Malcher et al., 2011, Schladebeck and Mösch, 2013).

In turn, Yak1p kinase controls the subcellular localization of several other proteins including

Msi1p (Pratt et al., 2007), Crf1p (Martin et al., 2004), Pop2p (Moriya et al. 2001) and Bcy1p

(Griffioen et al., 2001). Some of these proteins (like Crf1p) may impact growth rate, while others (like

Bcy1p, which is part of PKA) may have additional signal transduction or feedback functions. Active

Yak1p also phosphorylates several transcription factors, including Hsf1p, Msn2p (Lee et al., 2008) and

Haa1p (Malcher et al., 2011), which are key transcription factors that modulate a cell’s resistance to

various stresses as well as Phd1p that affects the adhesion of yeast cells for other yeast cells. Yak1p

can bind to the yeast proteins Bmh1p and Bmh2p (Moriya et al., 2001; Lee et al., 2011) which seems

to influence the kinase’s activity. Recently, Lee et al. showed that Yak1p autophosphorylates its own

N-terminal domain which influences the kinase’s activity (2011), but how it affects the activity

remains unclear.

Clearly, we’ve learned a lot about Yak1p in the last 24 years. But there are many areas that

remain poorly understood. One way to expand our knowledge of Yak1p is to find proteins that

physically associate with Yak1p in vivo. Over

the last decade, there have been several large-

scale experiments designed to identify

complexes of proteins found in living yeast

cells. Reading through some of these papers

reveals 171 different proteins that have been

reported to bind Yak1p! It is very likely that

some of these results aren’t really meaningful

and would fail to be confirmed by other

methods. I’ve scanned the list and identified

ten different proteins that I think should be

interesting as putative Yak1p binding partners.

Over the course of the term, each pair of

students will investigate one of these putative Yak1p-binding proteins.

Putative Yak1p-Interacting Proteins

Protein Reference

Bck1p Ptacek et al., 2005

Caf20p Graumann et al., 2004

Dcs2p Ho et al., 2002

Dia2p Ho et al., 2002

Fks1p Breitkreutz et al., 2010

Gdb1p Ho et al., 2002

Hek2p Hasegawa et al., 2008

Pop2p Moriya et al., 2001

Ybl055cp Fasolo et al., 2011

Ypl247cp Ho et al., 2002 and Breitkreutz et al., 2010

Saccharomyces cerevisiae

Baker’s yeast (Saccharomyces cerevisiae) is one of the simplest eukaryotic organisms and

needs a little introduction before we get farther with our project. Yeast are easy to grow,

nonpathogenic and their biochemical workings are well-established. Furthermore, it is easy to add or

delete DNA and the cells can grow as either a diploid or a haploid making genetic analysis very

accessible (think about how much easier BIO260 would have been if you were just working with

haploid flies!). Without a doubt, humanity understands the workings of a yeast cell better than any

other eukaryote (Botstein and Fink, 2011). However, most

scientists have absolutely no interest in the yeast itself; rather,

yeast serves as a model organism. By understanding

fundamental processes and proteins like Yak1p in yeast

(where the experiments are relatively easy), we can learn an

enormous amount about the biology of other eukaryotes, such

as humans where experiments can be extremely difficult

(Hartwell et al., 1997, Mager and Winderickx, 2005).

The genome of baker’s yeast consists of about 12Mbp

on 16 chromosomes and its full sequence was determined way

back in 1996. From the sequence, it is predicted that yeast has

approximately 6,607 genes. Despite the fact that over 50,000

scientific papers have been published on yeast in the last 40 years, we unambiguously know the

function of only about half of these genes! For about one-fourth of the genes, we don’t even have a

good guess about their function. The genes whose functions have not yet been firmly characterized are

referred to as anonymous ORFs (ORF stands for open reading frame which is a sequence of amino

acids that could conceivably be translated into a protein).

When a yeast gene is studied, it receives a three letter and one number name. The three letters

refer to the function of the gene (often cryptically) and the numbers are assigned sequentially. For

example, RAD17 is a gene that confers resistance to radiation. The protein that corresponds to this

gene is Rad17p. Notice that genes are always italicized but proteins are never italicized and instead are

followed with a “p”. rad17 refers to a deletion of the RAD17 gene and rad17-4 refers to a mutant

allele of the RAD17 gene. If a gene is written with all capital letters, it is a dominant allele of that

gene; conversely, if it is written in lower case, it is a genetically recessive allele. Sometimes, we insert

one gene into the middle of another to disrupt its function. This is shown with a double colon, such as

in the example of rad17::URA3 which shows that the URA3 gene has been inserted into the RAD17

gene. RAD24 is a different gene, but shares the same phenotype of resistance to radiation, such that

rad24 mutant yeasts are more sensitive to radiation. For a more complete discussion of yeast gene

and protein nomenclature, see http://www.yeastgenome.org/help/yeastGeneNomenclature.shtml.

Please note that correct formatting of genes and proteins is expected in your final paper.

Obviously, anonymous ORFs don’t have this kind of a name yet. Rather, all genes in the genome have

been assigned a systematic name such as YBR195c. In this case, the first letter stands for yeast (so all

yeast ORFs begin with a Y). The second letter designates the chromosome on which the gene is found,

where chromosome 1 = A, chromosome 2 = B, etc. The third letter indicates the arm of the

chromosome. You may recall that chromosomes have a long and a short arm on either side of the

centromere. In humans, the arms are named p and q; in yeast, the arms are named R and L for right

and left. All the genes on that arm of the chromosome are then numbered sequentially from the

centromere to the telomere. The three digit number refers to this number. Finally, the systematic

name ends with either a ‘w’ or a ‘c’ to indicate which strand of DNA is the coding strand. One strand

is arbitrarily assigned as the ‘Watson’ strand and the other strand is the ‘Crick’ strand. Therefore,

YBR195c is the 195th

gene on the right arm of yeast chromosome 2 and is encoded on the Crick strand.

Our Research Goals

Early in the term, you and your lab partner will select one gene from the list on the previous

page to become “Your Favorite Gene” (YFG) and “Your Favorite Protein” (YFP) for the term. You

will work with this gene/protein for all 10 weeks of the term to answer two key questions: 1) Does

Yak1p affect the subcellular localization of YFP? and 2) Does Yak1p affect the phenotypes of YFG?

A rough flowchart for our work is shown on the next page. If Yak1p affects either of these

phenotypes, that will be strong evidence that the interaction of Yak1p and YFP really does happen in

vivo and is physiologically important. It also suggests that looking for an interaction between human

DYRK1A and the human ortholog of YFP might tell us new and important information about

DYRK1A function.

In the first week, we’ll meet in the computer lab (Carnegie 211) to choose YFGs, begin with

some background reading, identify some useful phenotypes and design primers. After that, each lab

group can decide what experiments to do in which order. As the flowchart suggests, most groups will

likely PCR amplify YFG and start to figure out an assay for phenotypes associated with YFG. After

that, it’s very hard to predict exactly what we’ll be doing from week-to-week because it is probable

that we’ll encounter difficulties and need to try some new strategies. I fully expect that we’re going to

run into some serious problems this term. That’s ok – that’s how the real world works! When we hit

problems, we’ll work our way through them. Even though we’ll have to try some procedures two or

three (or more) times, I expect that everyone will have an impressive dataset for their final paper. I

strongly encourage you to read thoroughly entire lab manual to think about the project as a whole.

The nature of these experiments means that this lab will require a substantial time investment

outside of our scheduled time slot. However compared to BIO200 or BIO260, you should have to

spend much less time writing up your data outside of lab since there is really only one large lab report

due. Additionally, most weeks you are not required to be present during our lab time (with the major

exception of the first week). You are welcome to get the work done whenever it fits well in your

schedule and when your experiments need your attention. Be aware that BIO260 has lab scheduled on

Thursdays, so they have priority for the space and equipment during those times. Even with that, you

should be able to quietly slip in a corner and get your work done or we can find a different space to

work in.

Many of the basic methods that you’ll need for these experiments are described in the

following pages of this packet. You may have to adapt them as we learn what’s working and what

isn’t. You will also need a copy of the Biology Student Handbook as this has essential information on

basic tasks like cleaning glassware, sterile technique and spectrophotometers and gel electrophoresis. I

strongly encourage you to coordinate with other members of the lab to make media and other reagents

that can be shared. Additionally, there are a series of yeast references books that have been placed on

reserve in the library. Please know what you’re doing each day and come to lab prepared. Reading

things well in advance will help you avoid frustrations. For example, you may need to stop by and

start a culture the day before lab so that you’ll have some cells to work with during your lab time. If

you’re confused about something, please ask! Your professor is here to be your major resource to help

you on this project.

I expect that each student will be able to develop a very solid dataset to write your paper on

after ten weeks of work. You have certainly learned by now that things don’t always go well in lab. In

other classes, we just shrug and say that it’s too bad. In this class, you need to learn from your

mistakes and try again. Problems are certain to happen and we should have sufficient time to deal with

them.

Key Plasmids

We will be working with a few key plasmids during this term that need to be introduced. The

first is pYES2.1-GFP (Drinnenberg et al., 2009). The full sequence and documentation of this

plasmid is available at

http://www.addgene.org/22308

but I will introduce some key

features here. Most

importantly, this plasmid

contains a gene encoding for an

enhanced green fluorescent

protein (EGFP, which is a

mutant that fluoresces more

brightly than wildtype GFP).

Immediately in front of the

EGFP gene are restriction

enzyme sites for NotI and

XmaI. We will clone YFG

(minus its stop codon) into

these two sites so that yeast

cells will synthesize a single YFG-EGFP fusion protein. The sequence of a portion of this plasmid is

shown below with the restriction enzyme sites and the EGFP start codon highlighted. Since we want

YFG and EGFP to be a fusion protein, it is

essential that we maintain the translational

reading frame between the carboxy-

terminus of YFG and the amino-terminus of

EGFP.

The resulting fusion protein will be under the control of the promoter from the GAL1 gene of

yeast. This promoter is strongly induced by the presence of the carbohydrate galactose and is strongly

repressed by the presence of glucose. Thus, we should be able to “turn on” and “turn off” this

promoter by simply changing which carbohydrates the yeast cells receive. The plasmid also contains

an origin of replication that is derived from the naturally occurring 2 plasmid. This plasmid is

maintained at a high copy number (about 40-50 plasmids per cell) so we should get plenty of fusion

protein produced. A colE1 origin and an ampicillin-resistance gene are also present so that we can

manipulate our plasmid in E. coli. Finally, the plasmid contains a URA3 gene. This gene encodes the

Ura3p enzyme which is required to synthesize the nucleotide uracil which is essential for life. Since

the yeast cells receiving this plasmid are ura3, they would normally die on media that doesn’t contain

uracil (in other words, SDC-ura media). When we transform our plasmids into these yeasts, they will

now be able to live on SDC-ura, thus providing a strong selection to identify our transformed yeast

strains.

A second key plasmid that we will use is pRS426-YAK1 (Zhang et al., 2001). This plasmid

allows for the overexpression of the YAK1 gene. This plasmid contains the full-length, wildtype YAK1

gene with its natural promoter. It also contains the 2 origin so that the plasmid will be present at a

high copy number, thus effectively overexpressing the YAK1 gene. It contains the colE1 origin and an

ampicillin-resistance gene to allow it to be maintained in bacteria and a URA3 gene so that we can

select for the transformation of the plasmid into ura3 strains of yeast.

The final plasmid that I anticipate we will work with is pyak1::LEU2, which was originally

named pGS136-A (Garrett and Broach, 1989). Rather than adding a gene to the cell, the goal of this

plasmid is to disrupt a gene that is present in the genome. The plasmid contains an origin and

ampicillin-resistance gene so that we can maintain it in E. coli but does not contain an origin of

replication that can be used in yeast. The key part of the plasmid is that it contains two small

fragments of the yeast YAK1 gene that are separated by the wildtype LEU2 gene. Our recipient cells

are leu2 and therefore unable to grow without the amino acid leucine (SDC-leu plates). When we

transform this DNA into yeast cells, it is possible to have two recombination events happening

between the genomic YAK1 gene and the small fragments of YAK1 on the plasmid. When this

happens, the genomic YAK1 gene is disrupted and is nonfunctional. In addition, the cells can now

grow on SDC-leu media due to the addition of a wildtype LEU2 gene. Because homologous

recombination happens more frequently with linear DNA (compared with circular plasmid DNA), we

will cut this pyak1::LEU2 plasmid with the restriction enzymes SmaI and HinDIII before transforming

it into our yeast strains.

Learning About YFG

“A month at the lab bench can save you a day in the library”

With very few exceptions, it is always best to begin a new project by reading. There is a

massive amount of research that has been done on YAK1 and YFG so it would be crazy to ignore it.

Most of the high-quality research that has been done gets published in the peer-reviewed literature.

There are many databases available to you through the library’s website to search the world’s

information and none of them are truly comprehensive, covering

all knowledge in the world. The one key database that does

cover all biomedical research that we are interested in is

PubMed. This free database is part of the National Library of

Medicine, which is part of the National Institutes of Health and can easily be accessed at pubmed.gov.

To make the most of this essential tool, I have a few suggestions:

If you’re overwhelmed with information and don’t know where to begin, look at review articles

first to build a basic understanding of the topic or gene you’re researching.

Combine search terms. Use “AND” (in all caps) to search on two key terms at once.

Use the tag [ti] to search for terms that are found in the title. Without this tag, you will get

papers with your keyword in either the title or the abstract.

Narrow your results by using the filters on the left side of the screen. Particularly useful filters

are to ask for Review articles only, or to find only recent articles or to find articles that are

available in free, full-text. Notice that the free, full-text articles are available to everyone for

free – NCC has subscriptions to many journals that you can access for free that are excluded

from this list.

Saccharomyces Genome Database

The peer-reviewed, published literature isn’t the only place to find useful information. Of

course, peer-reviewed work is the gold-standard for high-quality scientific information, but it can often

be difficult to wade through many papers to find the

bit of information that you need. Fortunately, there

are some tools available to help sort through the vast

amount of data available. When looking at yeast genes and proteins, I strongly recommend the

Saccharomyces Genome Database (SGD) located at www.yeastgenome.org. This is a free database

supported by the U.S. National Institutes of Health. There is a full-time staff of about twenty

maintaining and improving the database but the actual information comes mostly from the peer-

reviewed published literature, supplemented by some information from scientific meetings or is

directly contributed yeast scientists around the world. Thus, SGD is a great tool for summarizing lots

of high-quality information in an efficient fashion. But don’t use SGD as your final stop! Nothing on

SGD should be cited in a lab report. Rather, use this database as a way to find information that you

need and then go to the published literature (which SGD has cited, with links) to flesh out the details.

To learn more about a specific yeast gene, go to the SGD homepage and type the gene’s name

into the search box in the upper left hand corner. This could be either the systematic (YJL141c) or the

common name (YAK1) of your favorite gene. This will take you to the “Locus Page” page for that

gene, which shows basic information about that gene and connects to a great deal more information.

Near the top of the page are a few sentences that describe the gene’s function, which can make a

reasonable first introduction.

The usefulness of SGD certainly doesn’t end with this short description. Indeed, there are links

from the locus page to a wealth of information about this gene. The “Interactions” tab will show what

proteins have been reported to bind to your favorite protein and is the tool that I used to generate the

list of potential YFGs in the Background section of this booklet. You can retrieve and analyze protein

and gene sequences, find what transcription factors are predicted to regulate this gene, find how the

transcription of this gene changes in response to many different environmental or genetic conditions.

You can align the gene or the protein sequence with its nearest neighbors from humans, from other

fungi or other model organisms. You can see biochemical pathways that metabolic enzymes

participate in. You can find other proteins that this protein is known to associate with. You can search

the yeast literature that SGD has curated. In many ways, this is a less powerful search than a PubMed

search as it is more restricted. However, the ‘textpresso’ tool can be a very helpful one. If you do a

PubMed search for “YAK1”, it will show you all the papers that have been published with “YAK1” in

the title or the abstract. But if YAK1 is mentioned in one paragraph in the discussion, it will be missed

by PubMed. A textpresso search will search the full-text of every SGD-curated paper and thus may

pull up many more obscure hits.

In addition to being a good introduction to YAK1 and YFG with links to key references, you

will need to gather at least two specific pieces of information about YFG beginning with this database.

One of your goals is to identify a phenotype of yfg that you can readily measure. To begin, I

recommend that you scroll down the Locus Page to the Phenotype section. Notice that it lists the most

basic phenotype for the null (or complete deletion) allele of YFG, showing that the cells is viable.

Thus, loss of YFG doesn’t result in the death of the cell (which is true for about two-thirds of yeast

genes). It also briefly lists several additional phenotypes. Click on the “All YFG phenotypes” link on

the right to go to a new page with much more information. Scan through the listed information and

begin to find phenotypes that we can easily measure. In most cases, we are looking for some condition

where yfg cells die but wildtype cells live (or vice versa). For example, are yfg cells more resistant

to high concentrations of zinc? Or are yfg cells more sensitive to a cell cycle inhibitor? See “Serial

Dilutions to Quantify Growth” section in this booklet to get a better understanding of how we will

likely measure the impact of YAK1 on the phenotype of yfg cells. Be aware that just looking at the

list of phenotypes on the SGD site will not give you nearly enough information to have some

confidence about which phenotype to measure. Use the links on the right side of the page to pull up

the paper where the actual phenotype was measured. That paper (or papers) will be crucial since this

booklet doesn’t list a precise protocol for measuring the phenotype of yfg cells! Rather, you will need

to extract the crucial information for the primary literature to figure out how to measure this

phenotype. That’s part of the educational experience of this lab.

A second goal is to identify the DNA sequence of YFG to help you design PCR primers. From

the Locus Page, retrieve the full genomic sequence of YFG. Our objective is to use PCR to amplify

this sequence from a wildtype yeast genome so that we can put it in a plasmid where it will be

translationally fused to the gene for GFP. pYES2.1-GFP (Drinnenberg et al., 2009) is the plasmid that

will receive your favorite gene and was described earlier in this booklet. The plan is to clone YFG into

this plasmid through the NotI site (to be introduced at the beginning of YFG) and XmaI site (to be

introduced at the end of YFG). These restriction enzyme recognition sites will be introduced through

the PCR primers that we design. However, if YFG contains a NotI site (GCGGCCGC) or XmaI site

(CCCGGG), this strategy won’t work, since the restriction enzyme will cut the middle of your gene.

When you have the full sequence of YFG from the database, confirm that it lacks both of these sites. If

it has an XmaI site, we will put NotI sites on both of the PCR primers. In this case, we expect about

half of our cloned plasmids to have YFG in reverse orientation, so we’ll have to screen those out.

You and your partner need to design two PCR primers. The “forward” primer should have 20

nucleotides that exactly match the 5’ end of the YFG coding sequence, beginning with the start codon.

In addition, we will add on a “tail” at the 5’ end of this primer. The tail will contain a recognition site

for the NotI enzyme plus three additional nucleotides (since restriction enzymes sometimes cut less

efficiently if they are located at the very end of a DNA molecule).

The “reverse” primer should have 20 nucleotides that exactly match the 3’ end of the YFG

coding sequence, not including the stop codon. The tail on the 5’ end of this primer should include a

site for XmaI with three additional nucleotides. This primer also needs to bind to the opposite strand

of DNA as compared with the forward primer. Finally, watch the translational reading frame!

Remember that we are aiming to build a fusion protein so we need to keep YFG and EGFP in the same

reading frame. Document what you’re doing by taking good notes in your notebook.

Once you have designed your two primers, email their sequences to me. I will verify them and

then order our primers so that we can proceed to PCR.

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