MARK 3 [6His-tagged]


Catalogue Number
66-0004-050
Product Size
50 µg
Price £
£330
Accession Number
AAC15093.1
Residues Expressed
2-729
Certificate of Analysis Size
50 μg
Species
Human
Source
Sf21 insect cell-baculovirus expression
Quantity
50 µg
Storage
-70°C
Concentration
1 mg/ml
Formulation
50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 150 mM NaCl, 270 mM sucrose, 0.03% Brij, 0.1% b-Mercapto-ethanol, 1 mM Benzamidine, 0.2 mM PMSF
Molecular Weight
~86.1 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: AAC15093.1. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

Activity Assay: The specific activity of His-MARK3 was determined using the method described by Hastie et al. (2006) with the enzyme being assayed at several concentrations. His-MARK3 was incubated for 10 minutes at 30°C in kinase reaction buffer in the presence of CHKtide substrate (300 µM) and [γ-32P]ATP (100 µM). Duplicate reactions were stopped by spotting the assay mixture onto Whatman P81 paper - capturing the phosphorylated substrate. The radioactivity incorporated was measured on a scintillation counter and the enzyme's mean specific activity was calculated. 

His-MARK3 specific activity: 282.0 Units/mg (282.0 Units/ml)

1 Unit = 1 nmole of phosphate incorporated into the substrate in 1 minute

Substrate: CHKtide (KKKVSRSGLYRSPSMPENLNRPR)


Background

Protein ubiquitylation and protein phosphorylation are the two major mechanisms that regulate the functions of proteins in eukaryotic cells. However, these different posttranslational modifications do not operate independently of one another, but are frequently interlinked to enable biological processes to be controlled in a more complex and sophisticated manner. Studying how protein phosphorylation events control the ubiquitin system and how ubiquitylation regulates protein phosphorylation has become a focal point of the study of cell regulation and human disease. Cloning of human Microtubule Affinity Regulating Kinase 3 (MARK3) was first described by Peng et al. (1998). MARK3 is a member of the subfamily of protein kinases that include the AMP-activated protein kinase (AMPK) and, like AMPK itself, is activated by the tumour suppressor kinase LKB1 (Lizcano et al., 2004). The physiological roles of the MARK subfamily of protein kinases include the phosphorylation of microtubule associated proteins and the regulation of cell polarity. Members of the MARK sub-family also phosphorylate tau at sites that induce its dissociation from tubulin. Enhanced phosphorylation of these sites is an early hall mark of Alzeheimer's disease and is followed by abnormal aggregation of tau to paired helical filaments that are found in people with Alzheimer's disease (Marx et al., 2010). MARK3 contains a ubiquitin-like domain adjacent to the kinase catalytic domain and, similar to MARK4, undergoes Lys29/Lys33-linked polyubiquitylation that may inhibit its activity. It may also interact with the deubiquitylase USP9X (Al-Hakim et al., 2008).


References

Al-Hakim AK, Zagorska A, Chapman L, Deak M, Peggie M, Alessi DR (2008) Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains. Biochem J 411, 249-60.

Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1, 968-71.

Lizcano JM, Goransson O, et al. (2004) LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23, 833-43.

Marx A, Nugoor C, Panneerselvam S, Mandelkow E (2010) Structure and function of polarity-inducing kinase family MARK/Par-1 within the branch of AMPK/Snf1-related kinases. FASEB J 24, 1637-48.

Peng CY, Graves PR, Ogg S, Thoma RS, Byrnes MJ, 3rd, Wu Z, Stephenson MT, Piwnica-Worms H (1998) C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding. Cell Growth Differ 9, 197-208.

Background kindly written by:

Sir Philip Cohen FRS, FRSE

University of Dundee

Director of the Medical Research Council Protein Phosphorylation Unit (1990-2012)

Director of the Scottish Institute for Cell Signalling incorporating the Protein Ubiquitylation Unit (2008-2012)

Co-Director of the Division of Signal Transduction Therapy (1998-2012)

Deputy Director of the Division of Signal Transduction Therapy (from July 2012)

Professor Cohen's research group is studying the interplay between protein phosphorylation and protein ubiquitylation in the regulation of innate immunity.