Identification of kinases, phosphatases, and phosphorylation sites in human and porcine spermatozoa

Figures & data

Figure 1. Kinetworks™ Protein Kinase Screen (KPKS) of porcine spermatozoa. Known proteins are marked with an arrow and labeled with a ‘p’ for porcine and a corresponding number which can be used with the key for identification. A: proteins p1-p18 and B: proteins p19-p28.

Table 1. Kinases in porcine spermatozoa.

ID	Lane	Band	Protein	Key	MW	Qty
p1	2	1	v-raf sarcoma viral oncogene B1	RAFB	99	1.5
p2	2	2	Extracellular regulated kinase 3 (56)	ERK3	55	9.6
p2	2	3	Extracellular regulated kinase 3 (52)	ERK3	52	9.3
p3	3	1	Protein kinase B alpha	PKBa	60	3.9
p4	6	6	p38 Hog MAP kinase	p38	38	4.1
p5	6	7	Cyclin-dependent kinase 5	CDK5	23	5.0
p6	11	3	Protein kinase C lambda/iota	PKCl	60	5.7
p7	11	6	Stress activated protein kinase (JNK) (45)	SAPK	48	4.2
p7	11	8	Stress activated protein kinase (JNK) (38)	SAPK	40	1.2
p8	12	3	Extracellular-regulated MAP kinase 6	ERK6	47	6.4
p9	13	1	Protein kinase C zeta (88)	PKCz	87	7.0
p9	13	2	Protein kinase C zeta (78)	PKCz	80	10.2
p10	13	3	MAP Kinase Kinase 1 (MKK1)	MEK1	42	3.1
p11	14	2	p21 activated kinase 1	PAK1	64	4.8
p12	15	2	MAP kinase kinase 4	MKK4	38	2.5
p13	16	9	MAP Kinase Kinase 6	MKK6	31	0.8
p14	18	1	Glycogen synthase kinase 3 alpha	GSK3a	45	47.0
p15	18	2	Glycogen synthase kinase 3 beta	GSK3b	41	17.3
p16	19	2	Ribosomal S6 kinase 1	RSK1	75	9.0
p17	20	2	Ribosomal S6 kinase 2	RSK2	74	2.5
p18	20	6	Casein kinase 2 (39)	CK2	41	24.8
p18	20	7	Casein kinase 2 (37)	CK2	38	9.7
p18	20	8	Casein kinase 2 (35)	CK2	34	2.5
p19	3	9	Lymphocyte-specific kinase	LCK	44	4.2
p20	5	1	Death associated protein kinase 1	DAPK	160	1.0
p21	5	5	Oncogene Lyn	LYN	48	6.6
p22	10	3	Calmodulin-dependent kinase 4	CaMK4	62	5.7
p23	11	2	Calmodulin-dependent kinase 1	CaMK1	44	7.4
p24	12	2	Calmodulin-dependent kinase kinase	CaMKK	48	3.8
p25	13	1	NIMA-related kinase 2	NEK2	47	8.4
p26	15	4	Protein kinase A	PKA	37	15.7
p27	16	1	Focal adhesion kinase	FAK	127	5.0
p28	18	4	ZIP kinase	ZIP	45	16.8

ID: location of proteins on Figure 1. Lane: each gel is divided into lane numbers 1 to 21. Lanes 1 and 21 are used for the molecular weight standards. Band: identifies the location of each detected protein in each lane. Protein: common protein name, isoforms are denoted by (#). Key: common protein name abbreviation. MW: the molecular mass is the protein size in KDa as measured by its location on the gel. QTY: relative quantity for a protein in its respective lane.

Figure 2. Kinetworks™ Protein Kinase Screen (KPKS) of human spermatozoa. Known proteins are marked with an arrow and labeled with an ‘h’ for human and a corresponding number which can be used with the key for identification. A: proteins h1-23 and B: proteins h24-34. Results of Western blot: Lanes 1 and 2 were incubated with Ab1 and Lanes 3 and 4 were incubated with Ab2. Lanes 1 and 3 are extracts of HCT-8 cells used as positive controls. Lanes 2 and 4 are sperm extracts.

Table 2. Kinases in human spermatozoa.

ID	Lane	Band	Protein Name	KEY	MW	Qty
h1	2	1	v-raf murine sarcoma viral oncogene B1	RAFB	89	6.5
h2	2	2	Extracellular regulated kinase 3	ERK3	54	16.6
h3	3	1	Protein kinase B alpha	PKBa	59	5.1
h4	3	2	Extracellular regulated kinase 1	ERK1	40	15.4
h5	3	3	Extracellular regulated kinase 2	ERK2	38	19.9
h6	4	2	3-phosphoinositide dependent kinase 1	PDK1	52	1.2
h7	4	4	Extracellular regulated kinase 2	ERK2	38	7.8
h8	5	1	Protein kinase C mu	PKCm	117	2.3
h9	6	5	p38 Hog MAP kinase	p38	38	8.9
h10	6	7	Cyclin-dependent kinase 5	CDK5	23	3.3
h11	11	1	Protein kinase C lambda/iota	PKCl	61	6.5
h12	11	3	Stress activated protein kinase (JNK) (45)	SAPK	48	7.2
h12	11	4	Stress activated protein kinase (JNK) (38)	SAPK	40	1.0
h13	12	2	Protein kinase C theta	PKCt	77	6.5
h14	12	5	Extracellular-regulated MAP kinase 6	ERK6	46	2.8
h15	13	1	Protein kinase C zeta	PKCz	78	37.0
h16	13	5	MAP kinase kinase 1	MEK1	41	5.7
h17	14	6	MAP kinase kinase 2	MEK2	41	3.6
h18	15	1	MAP kinase kinase 4	MKK4	38	2.6
h19	16	1	MAP kinase kinase 6	MKK6	31	6.3
h20	18	1	Glycogen synthase kinase 3 alpha	GSK3a	45	15.7
h21	18	2	Glycogen synthase kinase 3 beta	GSK3b	40	7.6
h22	19	1	Ribosomal S6 kinase 1	RSK1	76	1.4
h23	20	3	Casein kinase 2 (39)	CK2	39	20.6
h23	20	4	Casein kinase 2 (37)	CK2	38	15.9
h23	20	5	Casein kinase 2 (35)	CK2	34	21.1
h24	5	1	Death associated protein kinase 1	DAPK	154	5.1
h25	5	4	Oncogene Lyn	LYN	48	1.5
h26	6	1	Spleen tyrosine kinase	SYK	68	4.2
h27	11	2	Calmodulin-dependent kinase 1	CaMK1	43	3.1
h28	12	1	Calmodulin-dependent kinase kinase	CaMKK	51	5.4
h29	13	1	NIMA-related kinase 2	NEK2	50	5.1
h30	14	2	Oncogene Raf1 (70)	RAF1	73	5.1
h30	14	3	Oncogene Raf 1 (61)	RAF1	66	3.3
h31	15	2	Protein kinase A	PKA	36	11.6
h32	16	1	Focal adhesion kinase	FAK	120	3.3
h33	17	1	G protein-coupled receptor kinase 2	GRK2	72	6.5
h34	18	2	ZIP kinase (48)	ZIP	44	3.3
h34	18	3	ZIP kinase (45)	ZIP	41	3.3

ID: location of proteins on Figure 2. Lane: each gel is divided into lane numbers 1 to 21. Lanes 1 and 21 are used for the molecular weight standards. Band: identifies the location of each detected protein in each lane. Protein: common protein name with isoforms denoted by (#). Key: common protein name abbreviation. MW: the molecular mass is the protein size in KDa as measured by its location on the gel. QTY: relative quantity for a protein in its respective lane.

Figure 3. Kinetworks™ Protein Phosphatase Screen (KPPS) phosphatase and Kinetworks™ Phosphosite Screen (KPSS) phosphorylation site screens of human spermatozoa. Known proteins are marked with an arrow and labeled with a ‘s’ for phosphatase or a ‘k’ for phosphorylation and a corresponding number which can be used with the Key for identification. A: phosphatases along with B: phosphorylated proteins are indicated.

Table 3. Results from phosphatase and phosphorylation site screens.

ID	Lane	Band	Protein Name	KEY	Type	MW
s1	2	1	Protein phosphatase 2A – A	PP2A/A	PSTP	48
s2	4	1	Protein phosphatase 2C – d isoform	PP2C/d	PSTP	45
s3	6	1	Protein phosphatase 2C – b isoform	PP2C/b	PSTP	47
s4	8	1	MAP kinase phosphatase 1	MKP-1	PTYP	39
s5	10	2	Protein phosphatase 1	PP1/C-a	PSTP	30
k1	3	4	Cyclin-dependent kinase 1	CDK1	Y15	26
k2	4	3	p38 alpha MAP kinase	p38	T180/Y182	38
k3	8	2	Extracellular regulated kinase 2	ERK2	T185/Y187	38
k4	17	7	Glycogen synthase kinase 3 alpha	GSK3a	Y279	44
k5	17	8	Glycogen synthase kinase 3 beta	GSK3b	Y216	38
k6	19	2	MAP kinase kinase 1/2	MEK1/2	S221/S225	39
k7	20	5	cAMP Response Element Binding Protein	CREB	S133	43

ID: location of phosphatases (s) or phosphorylation site (k) on Figure 3. Lane: each gel is divided into lane numbers 1 to 21. To conserve figure space, empty lanes were removed from the figure (Lanes 11–21 on A and Lanes 6–8 and 10–15 on B). Band: identifies the location of each detected protein in the lane. Key: PSTP: protein-serine/threonine phosphatase; PTYP: protein-threonine/tyrosine phosphatase. For the phosphorylation site screen the key refers to the epitope(s) of the target protein. MW: the molecular mass is the protein size in KDa as measured by its location on the gel. QTY: relative quantity for a protein in its respective lane.

Table 4. Comparison of protein expression patterns in human and porcine sperm.

Protein	ID	Human	Porcine
3-phosphoinositide dependent protein kinase	PDK1	1	1
Protein kinase B alpha	PKBa	1	2
Glycogen synthase kinase 3 alpha	GSK3a	21	79
Glycogen synthase kinase 3 beta	GSK3b	10	29
Calmodulin-dependent kinase 1	CaMK1	3	1
Calmodulin-dependent kinase 4	CaMK4		2
Calmodulin-dependent kinase kinase	CaMKK	4	1
Casein kinase 2	CK2 (35)	100	10
Casein kinase 2	CK2 (37)	75	39
Casein kinase 2	CK2 (39)	98	100
Cyclin-dependent kinase 5	CDK5	4	9
Extracellular regulated kinase 1	ERK1	4
Extracellular regulated kinase 2	ERK2	6
Extracellular regulated kinase 3	ERK3	18	16
Extracellular-regulated MAP kinase 6	ERK6	1	3
Focal adhesion kinase	FAK	5	2
G protein-coupled receptor kinase 2 (BARK2)	GRK2	8
MAP Kinase Kinase 1 (MKK1)	MEK1	4	1
MAP kinase kinase 2 (MKK2)	MEK2	3
MAP kinase kinase 4 (MKK4)	MKK4	6	8
MAP Kinase Kinase 6 (MKK6)	MKK6	2	2
NIMA (never in mitosis)-related kinase 2	NEK2	3	1
Oncogene Lyn	LYN	1	2
Oncogene Raf1	RAF1	12
p21 activated kinase 1	PAK1		3
p38 Hog MAP kinase	p38	11	7
Protein kinase A	PKA	22	6
Protein kinase C lambda/iota	PKCl	6	7
Protein kinase C mu	PKCm	2
Protein kinase C theta	PKCt	3
Protein kinase C zeta	PKCz	27	3
Ribosomal S6 kinase 1	RSK1	5	10
Ribosomal S6 kinase 2	RSK2		10
Stress activated protein kinase (JNK)	SAPK (45)	7	5
v-raf murine sarcoma viral oncogene homolog B1	RAFB	7	2
ZIP kinase (death associated protein kinase 3)	ZIP (45)	8	16

The trace quantity of a band in the Kinetworks™ Protein Kinase Screen (KPKS) as measured by the area under the intensity profile curve was normalized to 100. For human sperm, casein kinase CK2 (35) with a trace quantity of 37,474 and for porcine sperm, CK2 (39) with a trace quantity of 44,571 were normalized to 100. Because of the potential antibody differences and species sensitivity a quantitative comparison is not recommended.