Signal Tranducers and Activators Transcription (STAT) 5b Protein

as A Candidate of Growth Promoter in Broiler Chicken

Anwa Ma’ruf

1, 2

, Ratna Damayanti

and Nove Hidajati

Postgraduate School, Universitas Airlangga, Surabaya, Indonesia

Department of Basic Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga Surabaya, Indonesia

Keywords: STAT 5b, amino acid, growth, broiler

Abstract: The purpose of this study is to determine the molecular weight of the protein STAT-5b that exists in liver

tissue as a basis to determine the amino acid composition of broiler STAT protein phosphatase that

experiences growth due to increased growth hormone (GH). Broiler liver tissue samples isolated from

broilers were maintained for 21 days, then the examination was followed by SDS Page and Western Blott.

The results of Western Blott revealed that STAT 5b of 90 kDa had amino acid composition of

datnilvspvylypdip or aspartate, alanine, threonine, asparagine, isoleosine, leucine, valine, serine, proline,

valine, tyrosine, leucine, tyrosine, proline, aspartate, isoleucine, proline.

1 INTRODUCTION

Growth hormone has an important role in regulating

body growth and metabolism. GH metabolic effects

occur when GH receptors associate with and activate

tyrosine kinases. The bonding of GH to its receptor

can activate Janus Kinase 2 (JAK 2) and further

phosphorylate tyrosine in the JAK-2 GH-receptor

complex. This tyrosine then forms the bonding site

for a number of signalling proteins, such as signal

transducers and activators of transcription (STAT) to

induce the growth effect. STAT proteins that play a

role in providing healing signals are STAT 1, STAT

3, STAT 5a and STAT B. STAT proteins play a

significant role in regulating metabolic and growth

effects.

Increased growth in animal husbandry has great

implications and appeal to the world of poultry.

However, signalling of STAT protein and its

expression patterns in broilers during growth period

have not been identified. So, the identification of the

molecular weight and the composition of amino

acids protein signalling STAT in broilers during the

growing period due to increased GH can be used to

make synthetic STAT protein to spur the growth of

the broilers.

Until recently, it has only been known that the

weight of STAT1 protein molecules in broilers was

91 kD, while the weight of STAT 3 protein was 83

kD. Therefore, further research is needed to

determine the molecular weight of STAT 5b

proteins, as well as the amino acid composition of

STAT 5b proteins to be used as the basis for making

synthetic STAT proteins to stimulate broiler growth.

2 MATERIALS AND METHODS

The chickens were placed in a battery cage with a

capacity of one chicken per cage receiving feed

twice a day at 06.00 pm and 18.00 pm in an amount

of 10% less than standard. At the age of 21 days, the

chickens were cut to be sampled for their hepatic

tissue for the following tests: (1) Isolation of

signalling proteins STAT 5a and STAT 5b from

broiler liver tissue, (2) Analysis of STAT 5a and

STAT 5b protein signalling from broiler liver tissue

by using Dot Blot method and then continued with

SDS-PAGE (sodium dudecyl sulphate

polyacrylamide gel electrophoreses), (3)

Identification of molecular weight of signalling

protein STAT 5a and STAT 5b using Western Blot

technique by means of electrophoresis elucidated

protein from polyacrylamide gel, and (4)

Identification of amino acids by MALDI-TOF

method.

Ma’ruf, A., Damayanti, R. and Hidajati, N.

Signal Tranducers and Activators Transcription (STAT) 5b Protein as A Candidate of Growth Promoter in Broiler Chicken.

DOI: 10.5220/0007546205250527

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 525-527

ISBN: 978-989-758-348-3

525

3 RESULT AND DISCUSION

3.1 SDS Page for STAT protein of the

broilers’ liver

The result of SDS-PAGE of STAT protein of broiler

chicken liver tissue showed the presence of STAT

protein as in Figure 1.

Figure 1. SDS-PAGE of STAT protein from broilers’

The results of SDS-PAGE on broilers’ liver

tissue revealed the presence of STAT protein. The

SDS-PAGE results showed that there were several

bands visible. The markers 260 with 140 kDa, 140

with 100 kDa, and 100 kDa marker with 70 kDa

each portrayed one protein band. The protein band

formed between 100 kDa and 70 kDa markers was

suspected of STAT protein. The protein bands

formed in the liver were very clear, indicating that

the liver tissue illustrationed strongest antibody

antigen reaction.

Results of SDS-PAGE on the liver tissue

proteins showed the presence of protein bands

between 100 kDa and 70 kDa markers. These were

proteins with molecular weights of 90 kDa and 91

kDa. However, there has not been able to determine

whether these results were STAT 5a and STAT 5b

proteins or not because several other protein bands

were also formed between these markers. To prove

that the formation of protein band with molecular

weight of 91 kDa and 90 kDa was indeed STAT 5a

and STAT 5b, it was necessary to make further test

using Western blot test.

3.2 Western Blot for STAT 5b protein

The result of Western blot for STAT protein on

hepatic tissue showed the presence of STAT 5b

protein with a molecular weight of 90 kDa, as in

Figure 3.

Figure 2. Western Blot for STAT 5b protein from the

broilers’

The result of calculating the weight of the STAT

5b protein molecule revealed that its weight was 90

kDa. The formation of protein bands between 100

kDa and 70 kDa markers after calculation apparently

had molecular weight of 90 kDa. This suggested that

the SDS-PAGE protein tested with Western blot was

a STAT 5b protein from growing-phase broiler with

a molecular weight of 90 kDa. The formation of a

protein band with definite 90 kDa molecular weight

was due to a bonding between STAT 5b protein

resulting from SDS- PAGE and anti-phospho-STAT

5b(Ser731).

STAT 5b with molecular weight of 90 kDa in

hepatic tissue showed that in growing phase broilers

STAT 5a and STAT 5b proteins had almost the same

molecular weight. It is expected that by identifying

the weight of the protein molecules STAT 1, STAT

3, STAT 5a and STAT 5b in broilers, researcher will

be able to determine clearly the mechanism of

growth hormone in regulating growth and

metabolism.

3.3 Amino acid of the STAT 5b proteins

The results of the MALDI-TOP examination

revealed that the STAT 5b of 90 kDa had amino acid

composition of datnilvspvylypdip or aspartate,

alanine, threonine, asparagine, isoleosine, leucine,

valine, serine, proline, valine, tyrosine, leucine,

tyrosine, proline, aspartate, isoleucine, and proline.

ICPS 2018 - 2nd International Conference Postgraduate School

526

Growth hormone plays a role in regulating body

growth and composition (Foster, 1998). Growth

hormone has a significant biologic effect that is

influenced by insulin-like growth factor I (IGF-I) in

improving skeletal muscle growth (Younken, 2000).

Provision of in vivo growth factor in broilers led to

an increase in growth rate and muscle mass by 15%

and required 6.5% less feed than normal. This

increase in growth has great implications and appeal

to the world of poultry. However, the expression

pattern of growth factor gene during growth mass to

date has not been known clearly (Killefer, 2000).

STAT protein plays an important role in the

regulation of gene transcription by GH and other

cytokines that activate Janus Kinase (JAK). STAT

proteins originally identified in the signalling

interferon pathway (IFN) (Darnell et al., 1994) are

cytoplasmic factors that contain the SH-2 domain. In

the frequent tyrosyl-phosphorylation through–the-

JAK-kinase-initiated cocktail, the cytoplasmic

STAT protein forms a complex with another STAT

protein through the phosphorylated tyrosine

interaction of the SH-2 domain, trans-locates to the

nucleus, binds to DNA and then activates

transcription of the target gene (Ihle, 1996).

Growth hormone is known to activate STATs 1,

3, 5a and 5b. Tyrosyl phosphorylation of GH-

dependent STATs 1, 3, 5a and 5b are found in 3T3-

F442A fibroblasts, in the liver of mice with

hypophysectomy, in liver cell cultures and in various

over-expression systems. Tyrosyl phosphorylation of

STATs 5a and 5b are also found in human IM-9

cells and hepatic muscle as well as skeletal muscle

of normal mice (Smit et al., 1999)

.STAT1, also called P91, is identified as a

member of the factor 3 gene complex that is

stimulated by IFN (FU, 1992). GH signalling

analysis of JAK2 deficiency cells and mutated cells

in expressing GH receptors showed that activation of

GH-dependent STATs 1, 3, 5, and 5b requires

activation of JAK2 (Smit et al., 1997). This is

consistent with the finding that JAKs activation is

required for STAT activation (Muller et al., 1993).

JAK1 or JAK2 actively overexpressed in COS cells

will stimulate the binding of STAT1 to DNA

(Silvennoinen, 1993).

An indirect study has shown that GH stimulates

the phosphorylation of STATs 1, 3 and 5 in serine or

threonine in the liver. This phosphorylation will

increase DNA binding of STAT1, and STAT3 and

substantially alter DNA binding of STAT5 (Ram

et.al., 1996). STAT 1, 3, and 5a contain conserved

consensus sequences for phosphorylation of MAP

kinases and preliminary studies show that MAP

kinase is responsible for serum phosphorylation of

STAT1, STAT3 and STAT 5a. While STAT 5b does

not contain conserved consensus sequence,

phosphorylation is performed by other kinases other

than MAP kinase. Proteins STAT 1, 3, 5a and 5b

also contain protein kinase C and casein kinase for

phosphorylation process. This suggests that double

signalling pathways may converge on STAT

proteins for transcriptional activation by GH.

4 CONCLUSIONS

The weight of the STAT 5b of 90 kDa had amino

acid composition of datnilvspvylypdip or aspartate,

alanine, threonine, asparagine, isoleosine, leucine,

valine, serine, proline, valine, tyrosine, leucine,

tyrosine, proline, aspartate, isoleucine, and proline.

Identified amino acid sequence can be used as a

basis for making STAT synthetic proteins which are

expected to be used to extend the action or effects of

growth hormone. Increased effects of growth

hormone will spur livestock growth.

REFERENCES

Darnell J, Lodish H, Baltimora D., 1990. Molecular cel

biology. 2

Edition, New York: Scientific America

Books, p 715

Foster DN, Froudman JA, Harmon SA, Foster LK., 1998.

Baculovirus-mediated expression of chicken GH.

Tektran p 14

Ihle JN., 1996. Cell 84: 331-334

Kaiya H, Van Der Geyten S, Kojima M, Hosoda H,

Kitajima Y, Matsumoto M, Geeliissen S, Darras VM

and Kanagawa K. 2001. Chicken Ghrelin:

purification cDNA cloning, and biological activity.

Endocrinology 143: 3463

Killefer K, Kenny PB., 2000. Anim Vet Sci 1877.

Ram PA and Waxman DJ., 1999. J. Biol. Chem.

274:35553-35561

Ram PA, Park SH, Choi HK and Waxman DJ., 1996. J.

Biol. Chem. 271:5929-5940

Smit LS, Meyer DJ, Argetsinger LS et al., 1999

Handbooks of Physiology. Oxford University Press:

New York, pp445-480.

Younken RV, Zaou Y, Wang X et al., 2000. J Endocrinol

166:620-69

Signal Tranducers and Activators Transcription (STAT) 5b Protein as A Candidate of Growth Promoter in Broiler Chicken

527